The closures will affect lanes between Jarvis and Cherry streets as crews move work on the north portion of the highway to the southern lanes.
Officials said there will be no westbound traffic on the expressway between the Don Valley Parkway and Jarvis Street on Saturday.
Between 12:01 a.m. on Sunday and 5 a.m. on Monday, there will be no eastbound travel from Jarvis Street to the DVP.
As a result of the work, officials said some ActiveTO road closures will not be in effect this weekend.
The program, which closes roadways to give people more room to move around during the coronavirus pandemic, has been taking place every weekend recently.
However, because of construction work on the Gardiner Expressway, the closure of Lake Shore Boulevard East between Leslie Street and south of Woodbine Avenue, as well as Bayview Avenue from Front Street to Rosedale Valley Road, will be cancelled for Saturday and Sunday.
The closure of Lake Shore Boulevard West between Windermere Avenue and Stadium Road will still go ahead.