A businessman in India (Bill Murray) fails to catch his train as it pulls out of a station. He is beaten to it by a younger man, Peter Whitman (Adrien Brody), who is carrying heavy luggage. Peter reunites with his brothers Francis (Owen Wilson) and Jack (Jason Schwartzman) on the luxury train called "The Darjeeling Limited," which is traveling across India. The brothers have not seen each other since their father's funeral.
Francis, the oldest of the three brothers, has planned their journey in advance. The journey is supposed to culminate in a reunion with their mother (Anjelica Huston), but Francis tells his brothers that they are making the journey for spiritual self-discovery. He tells them of a motorcycle crash in which he was, explaining the bandages on his head. He reveals that he technically died in the incident, and wants to reconnect with his brothers. His brothers are not convinced of this, and get annoyed with Francis' controlling behaviors, a trait shown to be inherited from their mother. The three mistrust one another (Jack plans to leave the trip early, Peter is hiding the fact that his wife is pregnant). With his assistant Brendan's help, Francis draws up an itinerary for the trip and takes his brothers' passports to prevent them from getting off the train too early. The youngest Whitman, Jack, has written a short story which is similar to his own life, but he denies the similarities. He obsessively listens to the messages on his ex-girlfriend's answering machine at every train stop. Moreover, he has a fling with the train's stewardess Rita (Amara Karan), whom Francis nicknames "Sweet Lime" for the drinks she offers.
Peter, the middle brother, justifies his keeping many of his late father's possessions (his glasses, his keys) by claiming that he was their father's favorite. His wife, Alice (Camilla Rutherford), is expecting a baby, but Peter fears that their relationship may end in divorce.
In their trips through the Indian provinces, Francis has one of his loafers stolen by a shoe-shine boy; Peter buys a cobra, which later escapes from its transport container. This escape results in the brothers being confined to their cabins in the train. Francis and Peter get into a fight over the latter being their father's "favorite" and Jack uses pepper spray, trying to stop the fight. The train's Chief Steward (Waris Ahluwalia), whom the three brothers have repeatedly annoyed, throws the three of them off the train with all their luggage. Jack is sad to leave Rita, who did not want to carry on her relationship with him before. She asks him what is wrong with him, and he replies that he is "not sure" and will "tell her the next time he sees her". On their way back to civilization, the brothers see three young boys fall into a river while attempting to pull a raft across it. Jack and Francis rescue two of the boys, but Peter fails to save the third, who dies. This affects Peter deeply. In the boys' small village, where nobody really speaks English, the three brothers spend the night and are befriended by the villagers. They attend the boy's funeral.
In a flashback, the three brothers and Alice are in a car, going to their father's funeral. They stop to pick up their father's Porsche from the repair shop, but the car is not ready so the brothers leave. They find a suitcase of his things in the trunk. It is revealed that their father's death was a result of him getting hit by a car, and that their mother did not attend the funeral. During their time at the repair shop, the three brothers as they're grieving, get into a confrontation with a truck driver. Jack forgoes his father's funeral and impulsively skips town for Paris.
Back in the present, the Whitmans get on a bus, which takes them from the village to the airport. However, they rip up their tickets and decide to go visit their mother. She is a nun at a Christian abbey in India. The reunion is very emotional (it is learned that Francis's accident was, in fact, a suicide attempt) and the family is reunited for a time. The next morning, the three brothers find that their mother has left, after leaving the boys their breakfast. They find out that apparently, she "leaves for a while" sometimes.
On the way back the three brothers run for a train, and jettison all their baggage on the railway line as they and some porters run after the train. Jack reads his new short story, and gives in, accepting that it is representative of his own life. Francis wants to give the passports back to his brothers, but the brothers decide that the passports are safer with him.