The incident unfolded in the eastbound lanes of Highway 401, east of Colonel Talbot Road on Saturday night. Police were called to the scene just after 8:30 p.m.
Police say that a Chevrolet Equinox left the highway, ended up in a ditch and rolled.
Five people were in the vehicle at the time of the crash. Two of them were taken to hospital, one of whom later succumbed to his injuries.
An OPP news release says the driver was arrested at the scene and now faces "a number of Criminal Code charges."
The name of the driver and the passengers have not yet been released. Police say they are still in the process of notifying next of kin.
Anyone with information for investigators is asked to call police at 1-888-310-1122, or to call Crime Stoppers at 1-800-222-8477.