St. Joseph, Missouri, in 1859, is divided by a railroad track that separates the richer and poorer classes of people. From the richer side comes Ann Arnesen (Vera Ralston), daughter of Michael Arnesen (Rheinhold Schunzel), owner of the Pony Express. Michael hires Sam Cotton (Bill Elliott as William Elliott) to protect his pony line from hostile Indians and the attacks of the gang of Peter Marquette (Joseph Schildkraut), owner of a stagecoach line who fears losing his contracts to the pony riders. Sam finds himself in a difficult position because Michael's wife, Cathy (Gail Patrick), is in love with Marquette. Sam, despite several attacks by Marquette's men, organizes the pony line. The ailing Michael is shocked to death by his wife's confession of hate, and Marquette tries to destroy the ecpress stations. Sam, with the aid of a friendly Indian tribe, finally wipes out Marquette and his gang, and returns to St. Joseph and Ann, the woman he loves.

In 1859, bank president Michael Arnesen welcomes California Senator Gwin to his home in St. Joseph, Missouri, where he is hosting a party to introduce Gwin to his business and society friends. Attending the party are Arnesen's devoted daughter Ann and his second wife Cathy, who is having an affair with Peter Marquette, the owner of an influential stagecoach line. Cathy has warned Marquette of Arnesen and Gwin's plan to start an express mail service between St. Joseph and Sacramento, which will speed communication throughout the western states, and thereby hurt Marquette's business. Cathy assures Marquette that she will do anything to help him, regardless of her husband's interests. Arnesen takes his guests to a colorful saloon owned by gambler and cattleman Sam Cotten, who has just returned from a long cattle drive. Sam is dismayed to learn that Arnesen and the "reform crowd" have cleaned up the town so that the saloon will no longer be profitable, but is delighted to meet the lovely Ann and renew his friendship with Gwin, whom he has known since his days as a trader in the West. Sam sells the saloon and accepts Gwin's offer of a job with the new mail company, which is to be called the Pony Express. Marquette is angered when the new company refuses to rent his stations, which would force them to use a longer route, but in Washington, D.C., Cathy ensures that Marquette retains the mail franchise on which the new express company had bid. Arnesen is heartbroken that they have not obtained the franchise, but Sam assures him that if they build their own stations, they will be safe from Indian attack and able to charge large sums for private letters. Sam and his pal Dringo head the group scouting for station sites, and although Marquette hires gunslinger Feisty to kill him, Sam escapes his attack and continues working. Later, all of the stations have been established and Sam returns to St. Joseph to prepare for the opening festitivies. His bumbling friend, Judge Winters, misinterprets Sam's teasing comments about selling his company stock and almost ruins Sam's romance with Ann with some unfortunate comments. Meanwhile, determined to stop the Express, Marquette urges Cathy to kill her ailing husband. That night, Cathy's lurid revelations about her affair with Marquette cause Arnesen to die from a heart attack. When Cathy takes control of the bank soon afterward, she orders that the Express payroll not be met, which means the end of the company. Sam hears of her plans, however, and threatens to reveal her infidelity if she does not get out of town immediately. Cathy storms out, leaving Marquette to stop the Express on his own. Grand celebrations are held in St. Joseph and Sacramento on the day the first riders set out, and Sam and Dringo tour the stations. They are alarmed to learn that one of the stations has been burned, and when a survivor states that Indians are responsible, Sam, who is friendly with many tribes, investigates. Horseshoe prints at the site lead Sam to believe that white men disguised as Indians are the real culprits, and he also surmises that the dead Indian found at the scene was killed in advance by the outlaws and left as "proof" of the local tribe's guilt. Sam informs the dead man's father, tribal elder Wassac, then joins Wassac for peace talks to prevent him from leading his people in a war to avenge his son's death. When another station is attacked, Sam and the Indians arrive just in time to rout the outlaws, who are working under Marquette's orders. Sam is wounded in the fracas but chases Marquette, who, before succumbing to a fatal injury, begs Sam to find Cathy, who is alone in a mountain hideout. Sam goes to the cabin, where he is once again attacked by Feisty. Sam outdraws Feisty, but Cathy is killed by a ricocheting bullet. Ann and Dringo arrive, and Ann assures Sam that she is more interested in happiness with him than in the wealth generated by the Pony Express.