After witnessing gangster Eddie Kim brutally beat U.S. Prosecutor Daniel Hayes to death in Hawaii, Sean Jones is escorted by FBI agents Neville Flynn (Jackson) and John Sanders on a Boeing 747-400 to testify in a trial in Los Angeles. Despite increased security for the flight, Kim arranges for a time-release crate full of venomous snakes to be placed in the cargo hold in an attempt to bring down the plane before it reaches Los Angeles International Airport (LAX). To ensure the snakes attack the passengers without being provoked, he has one of his henchmen disguised as an airport ground employee spray the passengers' leis with a special pheromone which makes the snakes more aggressive in their attack.
The crate opens midway through the flight and the snakes make their way through the cabin. A cat in the cargo bay, a couple having sex in the bathroom, and a man using the bathroom are the first killed. The plane's captain, Sam McKeon, investigates and fixes an electrical short, but is killed by the viper that caused it. Co-pilot Rick, unaware of the snake, believes Sam has suffered a heart attack and continues toward LAX. Some of the snakes attack Rick, and while fending them off he accidentally releases the oxygen masks throughout the plane, causing several snakes to drop into the cabin with them. Numerous passengers, including Agent Sanders, are killed. The surviving passengers, who have made their way to the front of the plane, put up blockades of luggage in a desperate attempt to stop the snakes.
Rick is attacked and the plane starts to dip downwards, causing a food trolley to crash through the luggage blockade. The passengers flee to the upstairs first class cabin before blocking the stairwell with an inflatable liferaft. Agent Flynn and Flight Attendant Claire regain control of the plane. Rick retakes the controls and has Flynn go into the cargo hold to restore the air conditioning/ventilation system.
Agent Flynn contacts FBI Special Agent Hank Harris on the ground, who gets in touch with ophiologist Dr. Steven Price (Louiso). Based on pictures of the reptiles emailed to him via a mobile phone, Price believes a Los Angeles snake dealer known for illegally importing exotic and highly dangerous snakes to be responsible. After a shootout with the dealer, he reveals that Kim hired him to obtain the snakes. His supply of anti-venom is commandeered for the plane's victims, and Harris gives orders to have Eddie Kim arrested.
Harris contacts Flynn, telling him that anti-venom will be ready for the passengers when they land. However, Flynn discovers that the cockpit is filled with snakes and Rick is dead. After a brief discussion, Troy, Three Gs' bodyguard, agrees to land the plane based on prior experience. After everyone gets prepared, Flynn shoots out two windows with his pistol, causing the plane to depressurize. The snakes are blown out of the cockpit and the lower floor of the plane.
Flynn and Troy take the controls of the plane and Troy reveals that his flight experience was from a video game flight simulator. After an emergency landing, the plane makes it to the terminal. The passengers leave the plane and anti-venom is given to those who need it. Just as Flynn and Sean are about to disembark the plane, a final snake jumps out and bites Sean in the chest. Flynn draws his gun and shoots the snake, and paramedics rush to Sean, who is unharmed due to a bulletproof vest. As a token of gratitude, Sean later takes Flynn to Bali and teaches him how to surf.
- Samuel L. Jackson as Agent Neville Flynn, an FBI agent assigned to protect Sean Jones on his flight to Los Angeles.
- Julianna Margulies as Claire Miller, a flight attendant.
- Nathan Phillips as Sean Jones, a surfer and dirtbike racer who witnesses a brutal murder committed by Eddie Kim.
- Bobby Cannavale as Special Agent Hank Harris, Flynn's contact in Los Angeles.
- Rachel Blanchard as Mercedes, a socialite passenger who brings her pet Chihuahua Mary-Kate aboard.
- Flex Alexander as Clarence "Three Gs", a famous rapper who is germophobic and keeps a bottle of hand sanitizer with him and refuses to be touched by others.
- Kenan Thompson and Keith Dallas as Troy and Big Leroy, Clarence's bodyguards.
- Lin Shaye as Grace, the senior flight attendant who acts as the flight's purser.
- Terry Chen as Chen Leong, a martial artist who is among the surviving passengers.
- Elsa Pataky as Maria, a female passenger who brings her infant son aboard.
- Sunny Mabrey as Tiffany, a flight attendant who develops a crush on Sean.
- Tygh Runyan as Tyler, a very allergenic passenger.
- Emily Holmes as Ashley, Tyler's wife.
- Tom Butler as Capt. Sam McKeon, the captain of the plane.
- David Koechner as Rick, Capt. McKeon's co-pilot.
- Byron Lawson as Eddie Kim, a crime syndicate leader.
- Todd Louiso as Dr. Steven Price, a snake venom expert assigned by the FBI to communicate with Flynn.
- Scott Nicholson as Daniel Hayes, a U.S. Prosecutor who is murdered by Kim at the start of the film.
- Taylor Kitsch as Kyle "Crocodile" Cho, a man who is attacked by the snakes while having sex in the bathroom with his girlfriend.
- Samantha McLeod as Kelly, a girl that is attacked by the snakes while having sex in the bathroom with her boyfriend.
- Kevin McNulty as Emmett Bradley, an air traffic tower controller.
- Gerald Plunkett as Paul, a rude and arrogant British businessman who is unhappy with being moved to First Class and has a hatred of Americans, including dogs and infants.
The film gained a considerable amount of attention before its release, forming large fanbases online and becoming an Internet phenomenon, due to the film's title, casting, and premise. In response to the Internet fan base, New Line Cinema incorporated feedback from online users into its production, and added five days of reshooting. Before and after the film was released, it was parodied and alluded to on television shows and films, fan-made videos, video games, and various forms of literature.
Released in the United States and United Kingdom on August 18, 2006, the film received mixed to positive reviews with 68% of reviews positive and an average normalized score of 58%, according to the review aggregation websites Rotten Tomatoes and Metacritic, respectively. Despite the immense Internet buzz, the film's gross revenue did not live up to expectations, earning US$15.25 million in its opening weekend. The film grossed US$62 million worldwide before its release on home video on January 2, 2007.
- Samuel L. Jackson also appeared in other Die Hard scenario films including the 1995 sequel Die Hard with a Vengeance and the 1998 action-crime mystery film, The Negotiator.