The movie opens with outlaws Jack Colby (Lee Van Cleef), Ben Miller (Sheb Wooley), and Jim Pierce (Robert Wilke) meeting up on horseback and riding into the small town of Hadleyville on a Sunday morning. As church bells ring out, the townspeople eye the notorious gang warily. Meanwhile, Marshal Will Kane (Gary Cooper) and pacifist Quaker Amy Fowler (Grace Kelly) are getting married at the Justice of the Peace office. The three outlaws ride through town and settle in outside the train depot to await the noon arrival of gang leader Frank Miller.

Marshal Kane and his radiant bride are congratulated by the town mayor (Thomas Mitchell) and the retired Marshal (Lon Chaney, Jr.), and Kane reluctantly hangs up his star per his wife's wishes. Just then a telegram arrives announcing the pardon of Frank Miller, who Kane had arrested and sent to prison for murder years earlier. The station master declares that Miller is coming on the noon train. Along with the arrival of the other three outlaws, it is obvious that the Miller gang is reuniting to come after Kane and the judge who sentenced Miller. Kane and Amy are quickly hustled out of town in hopes of avoiding bloodshed. Once out on the open road, Kane suddenly turns his buckboard around and tells a bewildered Amy that he can't run from his past. Besides, the new Marshal won't arrive until the following day and the town will be defenseless. Back in town, the world-weary judge who sentenced Miller to prison is packing up his office and getting out of town. He advises Kane to do the same.

Kane's hot-headed young deputy, Harvey Pell (Lloyd Bridges), who was passed over for the Marshal's job, presses Kane to turn over the reins to him and get out of town. Kane declines. Complicating things is the fact that Deputy Pell has taken up with saloon owner Helen Ramirez (Katy Jurado), a former lover of both Kane and Frank Miller. With Miller coming back, Helen Ramirez decides to sell the saloon and leave town. Determined not to be a widow, Amy Fowler also buys a ticket on the departing noon train.

At the train depot, Ben Miller grows impatient and rides in to pay a visit to the town saloon, where he is welcomed like a returning hero. As Miller leaves, he runs straight into Kane and they exchange glares. Kane enters the unfriendly confines of the saloon to ask for volunteers and overhears the surly bartender speculating he will soon be shot dead. An angry Kane knocks him to the floor. The jeering saloon patrons rebuff Kane and he leaves empty handed. Kane then pays a visit to the packed church and interrupts the sermon to ask for help. A heated debate erupts among the parishioners, and a lesson in civics follows as a few brave citizens stand up for Kane and the rest find excuses to avoid conflict. The realization that he may be fighting alone begins to dawn on Kane. A visit to the retired and cynical former Marshal only produces another rejection.

Meanwhile, Amy drops in on a surprised Helen Ramirez, searching for an answer to Kane's stubborn refusal to leave. Amy thinks her husband is staying because of Ramirez. Ramirez sets her straight. Tired and stressed, Kane goes to the livery stable and thinks about saddling his horse and leaving. Deputy Pell sees him and follows him in, pressuring him to leave. When Kane resists, Harvey slugs him. A desperate fistfight ensues, which Kane wins. Kane goes to the barber's office to clean up and hears the undertaker building a coffin.

Back at the Marshal's office, the one man who has volunteered to stand with Kane backs out when he discovers they will be alone. After the man leaves, Kane makes out his will and leaves it in a desk drawer. The clock ticks down the last seconds before noon. Suddenly, the shrill whistle of the approaching train sounds. The three outlaws lace up their gunbelts as the train nears. In a heartwrenching scene, Kane steps out onto the street just as Amy and Helen Ramirez drive by on their way to the station. Amy looks away, but Helen doesn't. Just as the two women arrive at the station, Frank Miller steps off the train, makes eye contact with Helen, and turns away to strap on a gun belt. In an iconic scene, the camera goes wide to reveal Marshal Kane all alone on the deserted streets.

The Miller gang walks into town, where Kane is waiting. Kane catches a break when foolish Ben Miller smashes a shop window to steal a hat. Kane circles behind the gang on their blind side and calls out. When they whirl around, Kane drops Ben Miller. Amy hears the gunshot as the train pulls out and jumps off. She runs into town to find Ben Miller dead in the street. Kane is soon caught in a crossfire and takes refuge in the hayloft of the livery stable. Colby rushes the stable, only to be gunned down by Kane. The remaining two outlaws set fire to the stables and prepare to shoot Kane as he is smoked out. Kane drives the frantic horses out, concealing himself Indian-style in the midst of the fleeing horses. He escapes, but not without getting shot off his horse. The wounded Kane holes up in a small store as Miller and Pierce pour shots Into the building. Pierce stops to reload in front of the Marshal's office across the street and is suddenly shot in the back at close range. As he collapses, Amy is revealed behind the shattered window, gun in hand. Frank Miller realizes what has happened and takes Amy hostage, using her as a shield to approach Kane's position. Miller calls Kane out, threatening to kill Amy unless he shows himself. Kane lowers his gun and steps out. Knowing that Miller is about to kill her husband, Amy spins around and claws Miller's face as he throws her down. Marshal Kane quickly takes aim and kills Miller. As the townspeople flood into the street, Kane's wagon is driven up so the couple can resume their wedding journey. Will Kane surveys the ungrateful townspeople scornfully and drops his tin star into the dust.