The company, which owns the Maple Leafs, Raptors, Toronto FC and Marlies, announced his resignation Friday in a news release.
Anselmi first joined the organization in December 1996 and was appointed COO in 2004.
His resignation is the latest in a series of major changes at MLSE since new president and CEO Tim Leiweke took over June 30.
The company parted ways with Raptors boss Bryan Colangelo earlier this summer and Toronto FC president and general manager Kevin Payne was fired this week.
Anselmi's last day with the company will be Sept. 13.