An American businessman (Bill Murray) speeds through city streets of India in a taxi. He arrives at his train station, only to see the train, The Darjeeling Limited, pulling away. He frantically chases it, but eventually gives up and is overtaken by a younger man who manages to climb aboard.
The young man, Peter Whitman (Adrien Brody), is the second-oldest of three brothers who have not spoken in the year following their father's death in a car accident. The youngest, Jack (Jason Schwartzman), had been living in Europe and undergoing a rocky relationship with an ex-girlfriend (played by Natalie Portman in the short introductory film "The Hotel Chevalier"). The eldest, Francis (Owen Wilson) was severely injured in a recent motorcycle crash (eventually revealed to be a suicide attempt) and wears bandages on his face. Francis had summoned his brothers to reconcile and take a spiritual journey through India together. While the brothers initially act happy to see each other, their lack of trust is evident.
All three Whitmans dose heavily from Indian prescription pain medications throughout the film, though Francis uses his injuries as an excuse. The two younger brothers soon grow tired of Francis's overbearing and bossy demeanor, and Jack confides in Peter his plan to leave early and meet his ex-girlfriend in Italy. Peter reveals that his wife, Alice, is seven months pregnant, and he behaves anxiously when speaking of the situation.
Francis is annoyed that Peter has kept many of their deceased father's personal items (sunglasses, luggage, car keys, etc.). This pushes their distrust even further. Jack is still conflicted and stoically depressed about his ex-girlfriend. Francis and Peter share a brief moment of agreement, as neither of them approved of the girlfriend, and they attempt to help Jack move on. Jack, meanwhile, romances and befriends a beautiful stewardess named Rita (Amara Karan).
Francis has planned the trip to an obnoxiously detailed level. His personal assistant Brendan (Wally Wolodarsky) occupies a different compartment of the train and prints out the schedules and itineraries that Francis provided. Their first stop is a beautiful temple, which the brothers enter after shopping at the nearby market (Jack buys a tear-gas gun and Peter buys a small cobra in a box). As they attempt to pray inside the temple, Peter becomes annoyed with Francis and moves to a different altar. Jack, whom Peter had sworn to secrecy, tells Francis about Peter's upcoming child. Returning to the train, the three brothers finally talk openly together and Peter admits that he never planned to have children because he never expected his marriage to last; not because he does not love his wife, but because of his low self-esteem. They re-board the Darjeeling Limited and continue their journey.
The next morning, the brothers awaken and learn with horror that Peter's cobra had escaped its box. They cannot find it in the compartment and run screaming into the hallway, leaving the head steward (Walis Ahluwalia) to capture it. The snake is confiscated and the brothers are forbidden to leave their compartment for the rest of the train ride.
Sharing the small compartment, the brothers' tempers begin to escalate. Francis chastises Peter for taking their father's possessions, and Peter, enraged, throws a belt at Francis, drawing blood. The two begin to brawl, until Jack ends the fight by spraying his brothers with tear-gas. They abandon their room during the ruckus, and the head steward kicks them off at the next stop.
The brothers walk with their luggage down a rural road next to a river. They spot three boys ahead, crossing the stream on a rickety pulley-raft. The current swells, the raft capsizes, and the boys fall in. The Whitman brothers immediately spring into action, jumping into the water and attempting to save the boys. Francis and Jack haul two of the children to safety, but the third is torn from Peter's grasp by the current and is taken over a small waterfall. As Francis and Jack rush to them, Peter emerges from the river, bloodied and extremely shaken, with the lifeless boy in his arms. Devastated by the child's death, the Whitmans are lead to the nearby village by the two surviving boys. While the boy's family and village elders frantically tend to the body, Francis, Peter, and Jack are given clean clothes and food. The two boys had explained that the Whitmans had saved their lives, and that Peter had done everything he could to save the third child. The Whitmans prepare to leave by bus the next day, but, to their surprise, they are invited to stay for the boy's funeral.
As the funeral takes place, a flashback shows the three brothers the year before, on the day of their father's funeral. They, along with Peter's wife Alice (Camilla Rutherford), are en route to the funeral when Peter demands that they stop at the mechanic's and pick up their father's car, which was under repair. After much bickering and making themselves late for the funeral, they find that the car is still not working, and that their estranged mother will not be attending the funeral as promised. However, the brothers band together in their grief for the moment, reveling in the fact that they are all in the same situation for once.
The Whitmans decide to end their spiritual quest, and prepare to board a plane home. As they wait for their flight, Peter calls his wife on a payphone and reveals to his brothers that she found out that their child is a boy. Francis and Jack congratulate their brother, and Francis offers his $6,000 belt (a prop passed back and forth throughout the film) as an inheritance gift for his nephew. The brothers veto their trip home, and resolve to visit their mother, who is now a nun near the Himalayas.
Upon their arrival at the convent, they are greeted by their mother, Patricia (Anjelica Huston). She warns them that the area is dangerous due to a man-eating tiger that is loose nearby. As they settle in for the night, the brothers confront their mother and ask her why she did not appear at their father's funeral. She says she simply did not want to go, and that she was needed at the convent. Peter tells her about her upcoming grandchild, and they plan to continue their talk in the morning.
When the brothers awaken, they realize their mother had left the convent in the night (which, they are told, she often does). Realizing they will never be able to truly trust her, they begin to trust each other instead. They arrive at the station to catch a train, having decided to return home to America. The train pulls away as they arrive, and they chase after it, dropping their father's suitcases at they run and leaving the past behind. They board the train and happily head for home, as brothers once more.