Introduction: A majority of preschool-aged children spend a significant portion of every weekday in a preschool or child care setting, where they typically participate in limited physical activity. This study determined if an ecologic physical activity intervention in preschools increases children's moderate- to vigorous-intensity physical activity (MVPA). Design: RCT, with preschool as the unit of randomization and analysis. Child physical activity was measured by accelerometry. Mixed model analysis of covariance with preschool as a random variable was used to test the effects of the intervention on physical activity in the total group and in sex-specific subgroups. Data were collected in 2008-2010 and analyzed in 2012-2014. Setting/participants: Children in 4-year-olds' classrooms in 16 preschools, pair matched and assigned to intervention or control groups. Intervention: The intervention focused on increasing children's physical activity by changing instructional practices. Researchers trained preschool teachers to engage children in physical activity during (1) structured, teacher-led physical activity opportunities in the classroom; (2) structured and unstructured physical activity opportunities at recess; and (3) physical activity integrated into pre-academic lessons. Research staff encouraged teachers to adapt the intervention to their classrooms. Main outcome measures: Minutes/hour of MVPA during the preschool day. Results: In an analytic sample of 379 children (188 intervention, 191 control), those in the intervention schools engaged in significantly more MVPA than children in control schools (7.4 and 6.6 minutes/hour, respectively). This difference remained significant after adjusting for parent education and length of the school day (half versus full day). In the sex-specific analyses, the difference was significant for girls (6.8 vs 6.1 minutes/hour of MVPA, respectively) but not for boys (7.9 vs 7.2 minutes/hour, respectively). Conclusions: A flexible ecologic physical activity intervention that trains teachers to provide children with opportunities to be active throughout the school day increased MVPA in preschool children. (author abstract)