Objectives: A number of states have enacted regulations to increase physical activity in children attending child care, but most were not evaluated. In 2010, Massachusetts (MA) enacted a new regulation requiring 60 minutes of light, moderate, and vigorous physical activity (LMVPA) for children in child care; we conducted a prospective evaluation. We hypothesized that MA centers would comply with the regulation at follow-up, resulting in increases in children's LMVPA. Methods: We evaluated compliance with the regulation in MA using Rhode Island (RI) as the comparison. We measured physical activity in a longitudinal sample of 20 centers and cross-sectional samples of 180 children per state three times before and three times after the regulation took effect. We assessed physical activity using the Observation System for Recording Activity in Preschoolers. We conducted difference-in-differences tests to evaluate changes in LMVPA in MA compared with RI from baseline to follow-up. Results: Children were active for at least 60 minutes of LMVPA in over 80% of centers at baseline and follow-up in MA and RI. Nevertheless, LMVPA increased in both states. In multivariable adjusted regressions, LMVPA increased from baseline to follow-up [MA estimate 38.1 minutes; confidence interval (CI): 28.6, 47.5; p [is less-than or equal-to] 0.0001; and RI estimate 42.7 minutes; CI: 35.2, 50.1; p [is less-than or equal-to] 0.0001. The average difference-in-differences estimate indicated no difference in MA compared with RI (estimate -4.6 minutes; CI: -16.6, 7.5; p = 0.46) since LMVPA increased comparably in both states. Conclusions: Although LMVPA increased in MA, we observed similar changes in RI. Thus, other factors could have influenced children's physical activity. (author abstract)