Two thugs looking for a "George Kaplan" at a hotel bar see a waiter calling out for him at the same time advertising executive Roger Thornhill (Cary Grant) summons the waiter. Thornhill, thus, is mistaken for "George Kaplan". Kidnapped by the thugs, he is brought to the Long Island estate of Lester Townsend, and interrogated by spy Phillip Vandamm (James Mason). Thornhill vehemently denies he is Kaplan. Vandamm thinks he is lying and Vandamm's henchman Leonard (Martin Landau) tries to arrange Thornhill's death, but Thornhill manages to escape a staged drunken driving accident.
Thornhill fails to convince his mother and the police that he had been kidnapped and forcibly inebriated. Journeying to the scene of the crime with police, a woman at Townsend's home (Josephine Hutchinson), presumed to be Mrs. Townsend, says he showed up drunk at her dinner party. She says Townsend is a United Nations diplomat. While searching Kaplan's hotel room with his mother, Thornhill answers a phone call from the thugs who are in the hotel lobby. He escapes and visits the U.N. General Assembly building to meet Townsend. He discovers that Townsend (Philip Ober) is not the man he met on Long Island, and that Townsend is a widower. As Thornhill questions Townsend, one of the thugs throws a knife, hitting Townsend in the back, killing him. Thornhill catches Townsend as he falls and grabs the knife, giving the appearance that he murdered Townsend. Thornhill flees and attempts to find the real Kaplan.
Meanwhile, a government intelligence agency picks up the news and realizes Thornhill has been mistaken for "George Kaplan," a fictional persona created by the agency to thwart Vandamm. However, Thornhill is not rescued for fear of compromising their operation.
Thornhill sneaks onto the 20th Century Limited train. He meets Eve Kendall (Eva Marie Saint), who protects him from the police, but who, unbeknownst to Thornhill, is actually working with Vandamm and his thugs. In Chicago, Kendall tells Thornhill she has arranged a meeting with Kaplan at an isolated bus stop.
Thornhill waits, but no one comes. He is attacked by a crop duster plane, but steps in front of a speeding tank truck; the airplane crashes into the truck, and Thornhill escapes.
When he reaches Kaplan's hotel in Chicago, he discovers that Kaplan had checked out and left before Kendall said she talked to him on the phone. Thornhill goes to her room, but she leaves. He tracks her to an art auction, where he finds Vandamm and his thugs. Vandamm purchases a Mexican Purépecha statue and departs. To engineer an escape from the thugs, Thornhill disrupts the auction by acting erratically; the police are summoned and take him away. He tells them he is the fugitive murderer; the police release him to the government agency's chief Professor (Leo G. Carroll), who reveals that Kaplan does not exist, and was invented to distract Vandamm from the real government agent: Kendall. Thornhill agrees to help maintain her cover.
At the Mount Rushmore visitor center, Thornhill (as Kaplan) negotiates Vandamm's turnover of Kendall for her prosecution as a spy. "Kaplan" confronts Kendall; she shoots him "fatally" with a handgun (loaded with blanks), and flees. Thornhill and Kendall meet in a forest. Thornhill discovers Kendall must depart with Vandamm and Leonard on a plane. Thornhill evades the Professor's custody, and goes to Vandamm's house to rescue Kendall.
At the house, Thornhill overhears that the sculpture holds microfilm, and Leonard's discovery that the gun used by Kendall to kill Thornhill was filled with blanks. Vandamm indicates that he will kill Kendall during the flight. Thornhill tries to warn her, but he is captured. As Vandamm is boarding the plane, Kendall takes the sculpture and runs to Thornhill. They flee to the top of Mount Rushmore. They begin to climb down the mountain's sculpture, pursued by Vandamm's two thugs, Leonard and Valerian. After a harrowing chase, Valerian falls to his death, while Leonard is fatally shot by the Professor, who appears at the scene with park rangers.
Later, Thornhill invites Kendall, as the new Mrs. Thornhill, onto the upper berth of a train, which then, suggestively, enters a tunnel.