After darkness falls and the cool night air sets in, Carter mutters discontentedly about the cold-blooded posse they have joined: "Doin' this in the middle of the night's crazy...I got nothin' particular against hangin' a murderer and rustler, it's just, I don't like doin' it in the dark. There's always some crazy fool that loses his head and starts hangin' everybody in sight."

As the men ride up into the hills, a stagecoach on its way into town is stopped. Its passengers include a well-dressed San Francisco gentleman named Swanson and his recently-married wife Rose (Mary Beth Hughes), Carter's ex-girlfriend. Smith makes a ribald joke that causes "Ma" Grier to cackle loudly:

Smith: Did you just get married, Rose?
Rose: Just today.
Smith: No wonder you're in such a hurry.

After he notices Carter and Rose momentarily looking at each other, the prissy Swanson has a few polite and haughty words with Carter about the "impulsive" nature of his "new" wife, and his intention to possess her all for himself:

I'm pleased to regard any friend of my wife's a friend of my own. However, I don't need to remind you that the pleasure of such an acquaintance depends upon the recognition by all parties of the fact that Miss Mapen is now my wife. She must be given a little time to become accustomed to her new responsibilities. As yet, I must confess that I'm jealous of her least attention. You'll forgive me, I know. A bridegroom is prone to be overly susceptible for a time. Later, when we've had time to get accustomed to our new relations, I shall be delighted to welcome you and others of my wife's friends to our home in San Francisco if it is still a desire. Until then -

Later, the posse surrounds three exhausted, innocent homesteaders by their campfire in Ox-Bow Valley in the middle of the night, with a herd of fifty cattle grazing restlessly nearby:

• Donald Martin (Dana Andrews), a newly-arrived rancher and family man living at Pike's Hole
  
• Juan Martinez (Anthony Quinn), a stoic, defiant Mexican who feigns not speaking English
  
• Alva 'Dad' Hardwicke (Francis Ford, director John Ford's older brother), a senile, "feeble-minded" old man who is confused ("I didn't do it. I don't even got a gun...)

Doomed, Martin protests their innocence, calls their hearing a "farce," and demands a fair trial when they are accused of rustling and murder, but the demagogue Tetley argues back that they will conduct a rapid trial:

Martin: Even in this god-forsaken country, I've got a right to a trial.
Tetley: You're getting a trial with 28 of the only kind of judges murderers and rustlers get in what you call this god-forsaken country.
Cowboy: (while forming a noose from a rope in his hands) So far, the jury don't like your story.
Martin: I'm not gonna say another word without a proper hearing.
Ma Grier: Suit yourself, son, but this is all the hearin' you're likely to get short of the Last Judgment.

The evidence is circumstantial against them, but Farnley can only think only of bloodthirsty revenge as he creates one of the nooses ("cause the law's slow and careless around here sometimes, we're here to see it speeded up"):

• Fifty head of cattle with Kinkaid's brand are nearby
  
• There is no customary proof of bill of sale (Martin claims - "I bought 'em and paid hard cash for them...he (Kinkaid) was out on the range, he didn't have a bill of sale with him. He said he'd mail it to me") (Martin protests: "You can wait, can't ya, until you can see Kinkaid?")
  
• According to Farnley, he heard Kincaid tell him that he wasn't planning to sell any cattle this spring
  
• Martin tells the preposterous story that in Los Angeles the previous month, he bought a broken-down ranch (from Dave Baker) at Pike's Hole for $4,000 and moved in three days earlier with his wife and two children (Martin argues that they should check out his story: "Surely, it's not so far to Pike's Hole that you can't go over there and find out")
  
• Kinkaid's gun is in the Mexican's possession (he "found it...lying on the road")
  
• The old senile man "who invents things" fearfully blames the killing on the Mexican ("The Mexican did it. He told me so, no, I saw him do it...He was asleep and he didn't mean to tell me, but I was awakened, I heard him talkin' about it")
  
• The Mexican is identified as a wanted criminal (named Francisco Morez) with a record ("He was a gambler - they want him for murder")

With reason and justice as guiding principles, Davies defends Martin's assumption of innocence until proven guilty:

Listen men, I'm not trying to obstruct justice, but just as this young man says, this is a farce and it'd be murder if you carry it through. All he's asking is what every man is entitled to. A fair trial. You say you're innocent, Martin, and I for one, believe you.

He is quickly silenced by a punch from Deputy Sheriff Mapes. During the long night as the men are tried on the spot, time is deliberately prolonged to add suspense to the film (and to wait until daylight and the Sheriff's possible arrival). There are both arguments and counter-arguments about their fate. Martin asks for further investigation rather than a hurried trial and execution: "I'd do a lot of finding out before I'd risk hanging three men who might be innocent." Carter concurs:

Carter: If you got any doubts, Tetley, I say let's call off this party. Take him back to the Judge, like Davies wants.
Tetley: This isn't only slightly any of your business, my friend. Remember that.
Carter: Hanging's any man's business that's around.
Tetley: If your stomach for justice is cooling, Carter, I'd advise you to leave now before we proceed any further. Otherwise, your interruptions are gonna become very tiresome.
Carter: I still don't like it! Hangin' murderers is one thing, but to keep guys you don't know for sure did it standing around sweatin' while you shoot your mouth off, that's another.

At five minutes after three in the morning, Tetley arrogantly decides to give the guilty men a reprieve until dawn to write farewell letters, eat a meal and pray, after Martin pleads: "If you're human at all, you'll give me time to write a letter." Martin gives his letter to Davies for delivery to his wife, a "kind and understanding...beautiful" letter that isn't typical of a man who would kill and steal ("read it, and you'll know he's not the kind of man that would steal or kill"). When Davies starts to show the compassionate letter to Tetley to prove that his words prove his innocence, Martin demands that his letter remain private, even though its reading to the mob might dissuade them and save his life. With an ironic racial slur, Martin accuses Davies of being a turncoat 'white man':

It's enough to be hanged by a bunch of bullying outlaws without having your private thoughts handed around to 'em for a joke...I didn't write that letter to be passed around. It's none of these murderers' business...I thought there was one 'white man' among you, but I was wrong. Give me my letter.

Art judges that the deck is already stacked against the three homesteaders: "But all that kind of argument in the world can't stand up against branded cattle, and no bill of sale, and a dead man's gun."

Before the dawn's lynching, the "Mex" tries to break away and fires shots from Larry Kinkaid's gun. But he is shot in the leg in the attempt, apprehended, and brought back. While spitting indignant foreign epithets at his executioners, he digs the bullet out of his own leg with a knife, and proves to be able to speak fluent English. And he claims that he found the gun lying on the road.

After a tally that will determine the majority's decision "so there can be no question or mistaken reprisals," only seven of the 28 vote to stop the illegitimate proceedings. Against a "triple hanging," Davies and Sparks are joined by Carter, Croft, Tetley's son and a few others - they are the only ones for "putting this thing off and turning it over to the courts." The camera pans from left to right across the lustful, seedy, and nasty group of cowboys in the posse who vote to condemn the men and overrule the minority. The old man is so senile that he is unable to fully comprehend what is happening to him. As the Mexican gives his last rites confession in a shaft of light from the dawn, Carter cynically observes: "That must have been an awfully busy life."

The three men are led to the base of a gnarled tree for the hanging, where three nooses have been hanging prominently and ominously throughout the previous sequence. Gerald Tetley is ordered to participate by his father:

Gerald: I can't.
Major Tetley: We'll see to it that you can.
Carter: The kid's seen enough already. Why don't you let him alone?
Major Tetley: (to Carter) This is not your affair, Carter. Thank you just the same. (to his son) I'll have no female boys bearing my name. You'll do your part and say nothing more.

As their hands are tied behind their backs, Martin pleads with Major Tetley for reconsideration: "Justice? What do you care about justice? You don't even care whether you've got the right men or not. All you know is you've lost something and somebody's got to be punished...You butcher!" Carter attempts to defend them, but is restrained and silenced by the mob. The victims, with ropes around their necks, are placed on horses that are whipped out from underneath them. Gerald gets a vicious gun butt in the face from his father for refusing to whip one of the horses. The shadows of the three men's bodies are seen swinging on the ground. To "finish 'em," Farnley fires bullets from his rifle into all three men to ensure that they are dead. The posse leaves as Sparks sings about each of the three souls journeying through the Lonesome Valley and standing alone before their Maker.

On the way back to town, the posse meets Sheriff Risley (Willard Robertson) who is shocked to learn what they have done: "Larry Kinkaid's not dead." He explains that he just left a wounded Kinkaid being treated by a doctor at Pike's Hole and they "caught the fellas who shot him too." After Davies informs him that "all but seven" sentenced the three men to die, the Sheriff condemns them with contempt in his voice:

God better have mercy on ya. You won't get any from me.

Blame has to shift somewhere, so when the men return to town, Smith grumbles: "If you ask me, that Tetley is the one we ought to lynch." When Major Tetley returns to his home in town, his son accuses his father of taking delight in prolonging the three mens' agony before their deaths:

You loved it. That's why you kept them waiting so long. I saw your face, it was the face of a depraved murderous beast. There are only two things that have ever meant anything to you, power and cruelty. You can't feel pity. You can't even feel guilt. In your heart, you knew those men were innocent, yet you were cold - crazy to see them hanged....I could have stopped you with a gun just as any other animal can be stopped from killing, but I couldn't do it 'cause I'm a coward. Aren't you glad you made me go, father? Weren't you proud of me? How does it feel to have begot a weakling, Major Tetley? Does it make you afraid for the latent weakness in you too that other men might discover and whisper about?

To make up for his grievous error, the stiff-backed Major Tetley shoots himself (off-screen) behind his locked door.

In the film's conclusion, a group of shamed, morose people gather in the town's saloon after taking up a collection of donations for Martin's widow ("even Mapes chipped in"), totaling about $500. Art inappropriately remarks: "Not bad for a husband who don't know any better than to buy cattle in the spring without a bill of sale." His blunt words prompt Carter to read the letter that Martin has written, just before he was hanged. The poignant letter is a masterfully-written indictment of vigilante lawlessness - it is the film's most memorable scene:

My Dear Wife.
Mr. Davies will tell you what's happening here tonight. He's a good man, and he's done everything he can for me. I suppose there's some other good men here, too, only they don't seem to realize what they're doing. They're the ones I feel sorry for, 'cause it'll be over for me in a little while, but they'll have to go on rememberin' for the rest of their lives. A man just naturally can't take the law into his own hands and hang people without hurtin' everybody in the world, 'cause then he's just not breakin' one law, but all laws. Law is a lot more than words you put in a book, or judges or lawyers or sheriffs you hire to carry it out. It's everything people ever have found out about justice and what's right and wrong. It's the very conscience of humanity. There can't be any such thing as civilization unless people have a conscience, because if people touch God anywhere, where is it except through their conscience? And what is anybody's conscience except a little piece of the conscience of all men that ever lived? I guess that's all I've got to say except - kiss the babies for me and God bless you.
Your husband, Donald.

The listeners are shaken and remorseful, as the mournful harmonica tune Red River Valley rises on the soundtrack. Carter and Croft walk out of the saloon. With the film's last line, Carter expresses his desire to deliver the letter and help the widow of lynch-mob victim Martin:

He said he wanted his wife to get this letter, didn't he? He said there was nobody to look after the kids, didn't he?

They ride out of town the same way they rode in at the film's start (the same dog crosses back over the road for a symmetrical ending), to take the letter to Martin's widow.