The route serves to connect Waterloo, including its two universities, Kitchener and Cambridge to Milton, Mississauga and for further travel eastward, or to Guelph via Puslinch. Select trips on Fridays and Sundays provide express service from the two Waterloo universities to York University in the Toronto district of North York via stops in Brampton.
This route operates primarily on Highway 401, Highway 407 and Highway 403.
The 25 Waterloo-Mississauga route connects the University of Waterloo to Square One Bus Terminal, making stops at Wilfrid Laurier University, Kitchener Bus Terminal, Sportsworld, Cambridge SmartCentres, Aberfoyle Park and Ride and Milton Park and Ride/Carpool. During peak hours, additional buses connect to the Milton line at Milton GO instead of at Square One Bus Terminal. Throughout the school year, additional express buses provide direct service between the universities and Square One Bus Terminal, or to York University Bus Loop, making stops at the Hurontario Street/Highway 407 Park and Ride and Bramalea GO Station.
On August 30, 2014, a new 25F branch service was introduced for the 2014-2015 school year, operating select trips Fridays and Sundays from the Waterloo universities express to York University via eastbound Highway 407, following route 48 Guelph-York University service with similar stops.