It took OPP and firefighters several hours to round up the cattle on Highway 401 in Mississauga.
They got loose from a transport truck that struck a barrier approaching Hurontario Street around 11:30 p.m.
Provincial police were forced to close the express lanes in both directions.
Several animals were injured in the crash and had to be put down.
The driver was taken to hospital with what paramedics described as "moderate" injuries.
The closure lasted nearly seven hours.