We investigated the association between the angiotensin I-converting enzyme (ACE) gene insertion (I)/deletion (D) polymorphism and physical activity levels in boys at early pubertal stage (calendar age 12.04 ± 0.77 years). Body composition by DXA, pubertal stage and cardiovascular fitness on cycle ergometer were measured in addition to 7-day accelerometry. DNA was separated from the whole blood. Sedentary behaviour level was significantly lower in DD subjects compared to I allele carriers. A significant main effect of the D allele was found on total physical activity (F 1,256 = 5,453; p = 0.020; η (2) = 0.021] and on light physical activity (F 1,256 = 4.74; p = 0.030; η (2) = 0.018). Adding screen time as a covariate did not change ACE I/D polymorphism effect on total physical activity levels (F 2,256 = 3,326; p = 0.041; η (2) = 0.025). Carriers of the D allele had significantly higher light physical activity (F 1,256 = 4,710; p = 0.031; η (2) = 0.20), with screen time as covariate. In conclusion, ACE gene has a significant effect on sedentary, light and total physical activity levels in healthy 12-year-old boys.