It would ensure that full-day kindergarten classes with two instructors would have an average of 26 children.
It would also cap classes from junior kindergarten to Grade 3 at 23 students, with 90 per cent of classes at 20 or fewer students.
Grades 4 to 8 classes would have an average of 24.5 students, and Grades 9 to 12 would have an average of 22 students.
Education Minister Laurel Broten says the move will improve learning for students and keep teachers in the classroom.
The move comes as the government tries to convince teachers' unions to accept a wage freeze as it battles Ontario's $15-billion deficit.
Broten said the new regulation isn't an attempt to prevent teachers from trying to negotiate larger class sizes in exchange for enhancements to their contracts.
The new regulation "can guide ongoing dialogue" between the government and teachers, she said.
In April, Broten publicly chastised the Elementary Teachers' Federation of Ontario, which represents 76,000 workers, after the union walked away from a voluntary process to help guide upcoming negotiations.
ETFO has said that it will negotiate with individual school boards instead.
With teacher contracts set to expire in August, the provincial discussion table allows teacher and staff unions and trustee associations to discuss issues and come up with a framework for local collective bargaining.