According to the North Carolina State Highway Patrol, the crash happened around 2:41 a.m. near the 344-mile marker in the eastbound lanes.
Investigators say a red Chevrolet Camaro was traveling west in the eastbound lanes when it collided head-on with a black Honda Accord.
Both men inside the Camaro were pronounced dead at the scene. In the Honda Accord, the 14-year-old girl, who was in the passenger seat, also died at the scene, while the driver was airlifted to ECU Health Medical Center in Greenville with serious injuries.
I-40 eastbound was temporarily shut down just east of Exit 343 but has since reopened.
Troopers said they are still working to identify the victims and notify next of kin.