The Gardiner Expressway will be closed from Carlaw Avenue to the Humber Bridge. The DVP will be closed from Highway 401 all the way south to the Gardiner.
The closures extend from 2 a.m. to 2 p.m ET.
About 14,000 cyclists are expected to participate in the ride, which starts and finishes at Exhibition Place.
Last year's event raised nearly $6-million for heart and stroke research.