This study examined the effects of a childcare gardening intervention on children’s physical activity (PA). Eligible childcare centers were randomly assigned to: (1) garden intervention (n= 5; year 1); (2) waitlist control (n= 5; control year 1, intervention year 2); or (3) control (n= 5; year 2 only) groups. Across the two-year study, PA was measured for 3 days at four data collection periods using Actigraph GT3X+ accelerometers. The intervention comprised 6 raised fruit and vegetable garden beds and a gardening guide with age-appropriate learning activities. The sample included a total of 321 3–5-year-olds enrolled in childcare centers in Wake County, North Carolina, withn= 293 possessing PA data for at least one time point. The analyses employed repeated measures linear mixed models (SAS v 9.4 PROC MIXED), accounting for clustering of the children within the center and relevant covariates (e.g., cohort, weather, outside days, accelerometer wear). A significant intervention effect was found for MVPA (p< 0.0001) and SED minutes (p= 0.0004), with children at intervention centers acquiring approximately 6 min more MVPA and 14 min less sedentary time each day. The effects were moderated by sex and age, with a stronger impact for boys and the youngest children. The results suggest that childcare gardening has potential as a PA intervention.