At about 10:30 a.m. ET a westbound tractor-trailer became unhinged from its trailer and crossed the median into oncoming traffic, colliding with a car.
The highway was shut down for several hours in both directions between Cobourg and Grafton while Ontario Provincial Police investigated the collision.
The eastbound lanes of the 401 near Grafton were opened at about 3:30 p.m. on Monday, police said, but the westbound lanes are expected to be closed for several more hours.
The male tractor-trailer driver and the female driver of the passenger car were taken to hospital with non-life threatening injuries, OPP said.