Background: We examined longitudinal changes in children's physical activity during the school day, afterschool, and evening across fifth, sixth, and seventh grades. Methods: The analytical sample included children who had valid accelerometer data in fifth grade and at least one other time-point, and provided complete sociodemographic information (N = 768, 751, and 612 for the 3 time-periods studied). Accelerometer-derived total physical activity (TPA) and moderate-to-vigorous physical activity (MVPA) were expressed in minutes per hour for the school day (~7:45 am to 3:30 pm), afterschool (~2:25 to 6:00 pm), and evening (6:00 to 10:00 pm) periods. We used growth curve analyses to examine changes in TPA and MVPA. Results: School day TPA and MVPA declined significantly; we observed a greater decrease from fifth to sixth grades than from sixth to seventh grades. Afterschool TPA declined significantly, but MVPA increased significantly among girls and remained stable for boys. Evening TPA decreased significantly and MVPA declined significantly in girls and remained stable among boys. Conclusions: To inform the development of effective intervention strategies, research should focus on examining factors associated with the decline in physical activity during the transition from elementary to middle school, particularly during the hours when children are in school.