The 400-series highways are a network of controlled-access highways in the Canadian province of Ontario, forming a special subset of the provincial highway system. They are analogous to the Interstate Highway System in the United States or the Autoroute system of neighbouring Quebec, and are regulated by the Ministry of Transportation of Ontario (MTO). The 400-series designations were introduced in 1952, although Ontario had been constructing divided highways for two decades prior. Initially, only Highways 400, 401 and 402 were numbered; other designations followed in the subsequent decades. The network is situated almost entirely in Southern Ontario, although Highway 400 extends into the more remote northern portion of the province.

The Provincial Highway Network consists of all the roads in Ontario maintained by the Ministry of Transportation of Ontario (MTO), including those designated as part of the King's Highway, secondary highways, and tertiary roads. Components of the system—comprising 16,900 kilometres (10,500 mi) of roads and 2,880 bridges —range in scale from Highway 401, the busiest highway in North America, to unpaved forestry and mining access roads. The longest highway is nearly 2,000 kilometres (1,200 mi) long, while the shortest is less than a kilometre. Some roads are unsigned highways, lacking signage to indicate their maintenance by the MTO; these may be remnants of highways that are still under provincial control whose designations were decommissioned, roadway segments left over from realignment projects, or proposed highway corridors.