The film opens on a sleepy gas station, where the slack-jawed pump attendant (Charles Baker) is attacked by a seemingly rabid animal. Through a series of glimpses the attendant's body begins to contort after the attack. Meanwhile, a young couple, Seth Belzer (Paulo Costanzo) and Polly Watt (Jill Wagner), retreat to the wilderness for a romantic camping trip in the ancient forests of Oklahoma, but their plans are shattered when they are car-jacked by an escaped convict, Dennis Farell (Shea Whigham), and his drug-addict girlfriend, Lacey Belisle (Rachel Kerbs). As the foursome travel the back roads together, they get a flat tire when they run over a small splinter infected animal on the road. Finding shelter at the now abandoned gas station, they fend off larger incarnations of a parasite as it infects more victims.
Soon after their arrival at the gas station, Lacey discovers the infected still-living body of the attendant from the opening scene. Lacey is attacked and dies instantly, but her corpse slowly reanimates and becomes a new creature, which attacks the remaining survivors. While fighting this creature, Seth, Polly and Dennis discover that severed pieces of the infected victims are capable of attacking on their own. Sheriff Terry Frankel (Laurel Whitsett) arrives on the scene and attempts to arrest Dennis, but she is ripped in half by Lacey's corpse. The creature takes the top half of the officer's body to the roof and slowly bonds with it, creating a larger creature. Seth formulates a plan to call for help using the cop's radio left behind when her body was ripped in half. However, during the execution of this plan, the newly rebuilt creature attacks and takes the lower half of the cop. The trio hides in the gas station's walk-in refrigerator when a section of its arm manages to get in and attack them. It is then discovered that Dennis has been infected, as his left arm violently twists and twitches on its own. Seth and Polly amputate his arm to prevent the infection from spreading. Dennis later explains that he had been pricked by a splinter from a creature they encountered on the road. Seth also discovers that the creatures are actually just a mold or fungus taking control of the corpses they infect and consuming the blood, and, because it is a simple creature, they hunt based on temperature, attacking the warmest thing they can find.
By lowering his body temperature with bags of ice, Seth is able to sneak past the creature to the police car, while Polly and Farell distract it with fireworks. Seth makes it to the police car, but discovers that, without the keys, the car, and the police radio inside, are useless. Meanwhile, Seth's body temperature has risen, forcing Dennis to leave the gas station in order to lure the creature away from him. As a result, the creature manages to enter the gas station and Dennis and Polly are forced to hide in the freezer. Meanwhile, the discarded fireworks ignite the trail of flammable liquid left earlier and the gas station catches on fire. Seth is able to retrieve a shotgun from the police car and helps Polly and Dennis escape. Dennis offers to keep the creature at bay while Seth and Polly escape, and is infected in the process. He shoots one of the gas pumps with the shotgun and the creature is engulfed in flames. When the smoke clears, the creature is dead, but Dennis is still infected. Seth and Polly approach Dennis, who gives them a key to a bank account, telling them to give it to the wife of a man he shot and paralyzed who later died. Dennis shoots directly into a stack of propane tanks, incinerating himself, the gas station, and any remaining infected corpses.
Seth and Polly walk off into the sunrise, but the final scene reveals that there are other infected creature corpses lying dormant in the woods.
Splinter received generally positive reviews. On Review aggregator Rotten Tomatoes, it has a 74% rating based on 34 reviews with an average rating of 6.3/10, indicating "Fresh". Its consensus states "Never taking itself too seriously, Splinter scores as a fast-paced, fun thriller with more than enough scares." On Metacritic, it has a metascore of 58, indicating "Mixed or average reviews."
Splinter won six awards at the Screamfest Horror Film Festival: Best Editing, Best Score, Best Special Effects, Best Make-Up, Best Directing and Best Picture. Splinter was a nominee for Best Horror Film at the 35th Annual Saturn Awards, but it lost to Hellboy II: The Golden Army. It was also nominated in Spike TV's 2009 Scream Awards for Most Memorable Mutilation for the arm removal scene, but lost to Saw V's Pendulum Trap.