In the old West, a small frontier town is being controlled by ruthless mob boss Decker (Lyle Bettger) and his cronies. After the local sheriff dies under mysterious circumstances, Decker arranges to have the town drunk (Thomas Mitchell) appointed sheriff, thinking he will be ineffectual. But the new sheriff sends for Tom Destry, son of a famous two-fisted lawman, to be his deputy. When Tom (Audie Murphy) arrives, he isn't exactly the swaggering he-man the sheriff had in mind. In fact, Destry doesn't even carry a gun. But the new deputy's mild exterior masks a fierce determination to see justice done, as Decker and the other locals soon discover.

In the Western town of Restful, saloon owner Phil Decker signals his girl friend, singer Brandy, to help him win Henry Skinner's ranch in a crooked poker game. After Brandy secretly replaces one of Henry's aces with a two, he protests his loss but Decker's men beat him up. Outside, sheriff Joe Bailey assures Henry he will settle the matter. Decker, however, wants Henry's land to complete a block of holdings that will allow him to charge cattlemen high fees to drive their herds across the West, and when Bailey contests the game's outcome, Decker has his henchman, Curly, kill him. Decker then instructs Mayor Hiram J. Sellers to declare Bailey dead from a heart attack and to name town drunk Reginald T. Rags Barnaby as the new sheriff. Although the townspeople laugh at him, Rags immediately stops drinking and announces that he will send for Tom Destry, the son of legendary Sheriff Destry, to act as his deputy. Within days, Tom's stage arrives, but Rags is dismayed to note that Tom is small and refined, and further horrified when Decker forces Tom to admit that he carries no gun. In the saloon, Brandy beats Doc Curtis in poker, winning the hat of Curtis' wife Bessie Mae. When Bessie Mae finds out, she starts a fight with Brandy. Tom breaks up the fight by pouring water over the women, but then is run out of the saloon by Brandy, who furiously pelts him with glasses and furniture. With Tom now a laughingstock, Rags begs him to wear a gun. Tom explains that although he possesses his father's famous holster, he does not believe in guns and has cleaned up other towns without violence. When Rags refuses to deputize him, Tom prepares to leave, but after he sees Bailey's funeral procession and then learns that Rags has traded in his badge for a whiskey bottle, he asks Sellers to deputize him. Seeing this, Rags asks for his badge back. Their first task is to rescue Henry, his young son Eli, and his pretty niece, Martha Phillips, from Decker, who has arrived to claim the ranch. Tom states that he cannot lawfully keep Decker from taking over the ranch, prompting the citizens to believe that he is league with Decker. Quietly, however, Tom begins to investigate the poker game and Bailey's subsequent death. He visits Brandy, who is charmed by his assertion that he can see her softer side and admits that Bailey was murdered. Tom then queries Doc Curtis, threatening to jail him if he does not examine Bailey's body for the true cause of death. He then hears gunshots from the saloon, and the town watches skeptically as he and Rags confront Decker's ruffian henchmen. Tom calmly takes each man's gun and demonstrates his sharpshooting skills against the saloon wall. Shaken, the ruffians leave, after which Tom announces that, now that he has a bullet shell from each gun, he can compare them to any bullet found in Bailey's body. Soon after, a jealous Decker warns Brandy to stay away from Tom, but instead she finds the deputy and advises him that Decker plans to kill him. Their conversation is interrupted by cattleman Jack Larsen, who informs Tom that Decker is charging exorbitant amounts to drive cattle across his land. Mistrustful, Larsen insults Tom and Brandy, causing Tom to punch him. Later, while singing on the saloon stage, Brandy sees Decker's henchman surround Tom and stops the song in order to protect him. Just then, Doc arrives with the news that the bullet in Bailey's body came from Curly's gun, and Decker makes no protest as Tom arrests Curly. The next day, Tom also arrests Larsen, who has driven his cattle through Decker's gate. In the jailhouse, Tom almost convinces Curly to name Decker as his boss when Sellers announces they have chosen a jury to try him. Realizing the trial will be crooked, Tom refuses to allow it, then frees Larsen, urging him to keep his head until Decker has been dealt with lawfully. That night, after Eli informs Tom that Martha wants to kiss him, Tom is called into the saloon by Brandy, who urges him to run away with her. Gunshots then ring out from the jail, and Tom discovers that Rags has been killed and Curly sprung. Furious, Tom straps on his father's holster and heads back to the saloon, where Decker has encircled the bar with armed men. Tom sneaks in through a back door, and, from his hiding place, expertly shoots each of Decker's men. Just as the mayor shoots Tom in the hand, Larsen and Doc Curtis enter and shoot him. Decker takes aim at Tom, and when Brandy runs to protect Tom, Decker fires, shooting her in the back. Tom kills Decker and gathers Brandy in his arms, and she asks for a kiss but dies moments later. Days later, Tom and an adoring Eli stroll through town, collecting warm greetings from all of the citizens. They are shocked to hear gunfire in the now peaceful town, and Tom traces the noise to Martha, who is shooting randomly in order to gain his attention. Tom shuts the door to chastise her, but she pulls him into a kiss before he can protest.