Exactly one year after the explosion of Flight 180, college student Kimberly Corman is heading to Daytona Beach, Florida for spring break with her friends, Shaina McKlank, Dano Estevez, and Frankie Whitman. While waiting on the entrance ramp to Route 23, she has a premonition of a deadly pileup caused by a logging truck. She stalls her car on the entrance ramp, preventing several people from entering the highway, including lottery winner Evan Lewis, mother Nora Carpenter and her fifteen-year-old son Tim, businesswoman Kat Jennings, stoner Rory Peters, pregnant Isabella Hudson, high school teacher Eugene Dix, and Deputy Marshal Thomas Burke. While Burke questions Kimberly, the pileup occurs, but Shaina, Dano, and Frankie are killed by a car carrier after Burke saves Kimberly at the last second.
After the survivors are questioned at the police station, Evan is fatally impaled by a fire escape ladder while attempting to escape from a fire in his apartment. Aware of Death's presence, Kimberly seeks help from Clear Rivers, the last survivor of Flight 180 who committed herself to a psychiatric ward for protection after Alex Browning was killed by a falling brick. When Kimberly informs Clear that Evan was the first of the highway survivors to die, unlike in her premonition, Clear realizes that the survivors are dying in reverse order. Meanwhile, Tim is crushed by a windowpane while leaving the dentist with his mother. Clear decides to help and introduces Kimberly and Burke to William Bludworth, who tells them that only new life can defeat Death. Believing that the birth of Isabella's baby would foil Death's plan, Burke sends fellow marshal Steve Adams to take her into custody while he gathers the other survivors in his apartment and explains Death's plan to them. When Nora attempts to leave via an elevator, a chain of accidents results in her head becoming trapped in the doors, and she is decapitated.
The survivors take Kat's SUV to track down Isabella, who has gone into labor, prompting Adams to rush her to the hospital. Along the way, the survivors realize that the demises of the Flight 180 survivors affected all of their lives even before the highway pileup by saving them from prior deaths, which explains why Death is working backwards this time. The SUV then suffers a blowout, causing them to swerve into a stack of PVC pipes in a farm that penetrate the car and injure Eugene. Rescue workers arrive and assist the farm owners, the Gibbons family, with rescuing the others while Eugene is hospitalized. Using the Jaws of Life, Kat's rescuer accidentally activates her airbag, causing her head to be impaled on a pipe protruding from her headrest. Her cigarette falls onto a gasoline leak from a news van that explodes, launching a barbed wire fence into the air that dismembers Rory.
Guided by a vision of a doctor named Kalarjian who Kimberly believes will euthanize Isabella, Kimberly, Clear, and Burke rush to a hospital to save her, only to find Isabella and her baby unharmed. However, an explosion from an oxygen leak in Eugene's ward kills both Clear and Eugene. Kimberly realizes that Isabella was never meant to die at all, deducing that her visions of 'Isabella' were actually of herself. Kimberly remembers that only the creation of a 'new life' can defeat Death. She drives into a lake to drown herself, but is defibrillated and revived by Dr. Kalarjian, creating 'new life'.
Later, Kimberly and Burke have a picnic with the Gibbons and Kimberly's father. The Gibbonses explain that their son Brian was nearly hit by a news van after Rory saved him earlier. As a result, they witness Brian suddenly die in an explosion caused by a malfunctioning barbecue grill.
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