Purpose: Knowing that motor skills will not develop to their full potential without opportunities to practice in environments that are stimulating and supportive, we evaluated the effect of a physical activity (PA)-based intervention targeting childcare providers on fundamental movement skills (FMS) in preschoolers attending childcare centers. Methods: In this two-arm cluster-randomized controlled trial, six licensed childcare centers in Ottawa, Canada, were randomly allocated into one of two groups (three controls, n = 43; three interventions, n = 40). Participants were between the ages of 3 and 5 yr. Childcare providers in the experimental condition received two 3-h workshops and a training manual at program initiation aimed at increasing PA through active play and several in-center "booster" sessions throughout the 6-month intervention. Control childcare centers implemented their standard curriculum. FMS were measured at baseline and 6 months using the Test of Gross Motor Development-2. Results: Groups did not differ on sociodemographic variables. Compared with control, children in the intervention group demonstrated significantly greater improvement in their standardized gross motor quotient (score, 5.70; 95% confidence interval [95% CI], 0.74-10.67; P=0.025 and gross motor quotient percentile, 13.33; 95% CI, 2.17-24.49; P = 0.020). Over the 6-month study period, the intervention group showed a significantly greater increase in locomotor skills score (1.20; 95% CI, 0.18-2.22; P=0.022) than the control group. There was a significant decrease in the object control scores in the control group over the study period. Conclusions: A childcare provider-led PA-based intervention increased the FMS in preschoolers, driven by the change in locomotor skills. The childcare environment may represent a viable public health approach for promoting motor skill development to support future engagement in PA. (author abstract)