The film is centred almost entirely on the Compagnie Internationale des Wagons-Lits train the Rome Express, travelling between Paris and Rome. Just as the train is departing from a station in Paris, Zurta and his colleague Tony just manage to board the train. They have found out that someone they want to see is on board. Another passenger, McBain, a wealthy businessman travelling with his brow-beaten secretary/valet, Mills, learns that a painting by Van Dyck, which he had previously tried to buy and which had later been stolen, has not been recovered and says he would do anything to obtain it. Also on the train are an adulterous couple, an annoyingly sociable Englishman, a French police inspector, and an American film star who is tiring of her fame, accompanied by her manager/publicist. It transpires that the stolen painting is in the possession of a man, Poole, who conspicuously keeps his briefcase close to him at all times. When he agrees to join a poker game, he finds that one of the other players is Zurta, and Poole's reaction shows that they know each other. Poole is disconcerted and carelessly sets down his briefcase, which is later innocently taken by Mills, who has a similar briefcase. After the game ends, Zurta follows Poole to his compartment, forces his way in and confronts Poole, who offers to hand over the painting but finds he has the wrong briefcase. After Zurta threatens to throw him from the train, they struggle and Poole is killed. Meanwhile, McBain discovers in Mills' briefcase the stolen Van Dyck he had wanted to buy. When Poole's body is discovered by an attendant, the police inspector begins an investigation and interviews all those who have been in contact with Poole. Zurta learns that the briefcases have been switched and tries to recover it from McBain's compartment, but is apprehended by McBain and Mills until the inspector arrives. Then Mills discovers the stolen painting hidden in McBain's compartment and realizes that McBain found it. He tries to use it to blackmail McBain, but McBain outwits him and takes it to the police. As the police inspector begins to suspect him, Zurta leaps from the train in an attempt to escape, but is killed.
Like the post-war remake of this film, Sleeping Car to Trieste, details of the 'back story' of the film are few. Zurta appears to be a professional criminal who organised the art theft. McBain has previously attempted to buy the painting.
