Background: We compared differences in physical activity (PA) between pre/mid-pubertal and post-pubertal participants according to gender. Methods: The study included a total of 1842 healthy participants aged 12.5-17.4 years, who participated in the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study. Participants wore a uniaxial accelerometer (ActiGraph© GT1M, Pensacola, FL, USA) attached to their lower back for seven consecutive days to measure PA. Pubertal status was assessed by physical examination and the population was classified as pre/mid-pubertal (Tanner stages 1-3) or post-pubertal (Tanner stages 4-5). PA was compared between these groups according to gender during the whole week, on school-free days and on school days, before and after school, and during lessons and recesses. Results: When comparing the pre/mid-pubertal group with the post-pubertal group, girls' total PA did not differ between groups. However, a slight difference was observed in boys, among whom PA on school-free days showed a difference of 17.6% between the pre/mid-pubertal group and the post-pubertal group (679 kcounts vs 564 kcounts, respectively; P = 0.0007) and 20% (162 kcount vs 135 kcounts; P = 0.006) for school recess. There was no difference among girls. Conclusions: A reduced level of PA in the post-pubertal groups was only observed in boys during non-organized times such as on school-free days and during school recesses, with a moderate impact on total PA.