AIM: To examine: (i) if maturity-related gender differences in moderate-to-vigorous physical activity (MVPA) depend on how maturity status is defined and measured; and (ii) the influence of maturity level on compliance with PA recommendations. METHODS: The study involved 253 children (139 boys) aged 9.9 ± 0.9 years, with mean stature and weight of 1.39 ± 0.08 m and 35.8 ± 8.8 kg respectively. Their PA was evaluated using an Actigraph accelerometer (Model 7164). Maturity was assessed using the estimated age at peak height velocity (APHV) and a standardized APHV by gender (i.e. centred APHV). RESULTS: Boys engaged in significantly more MVPA than girls (P < 0.0001). There was a significant correlation between the centred APHV and MVPA in boys (r = 0.20; P = 0.016), but not in girls (r = 0.13; P = 0.155). An ancova controlling for the estimated APHV showed no significant interactions between gender and APHV, and the main effect of gender on MVPA was negated. Conversely, there was a significant main effect of APHV on MVPA (F 1,249 = 6.12; P = 0.014; η p (2) = 0.024). Only 9.1% of children met the PA recommendations, including 14.4% of boys and 2.6% of girls (P < 0.01). This observation also applies in both pre-APHV (12.7% of boys vs. 2.4% of girls, P < 0.001) and post-APHV children (23.8% of boys vs. 3.4% of girls, P < 0.0001). No differences in PA guidelines were observed between pre-APHV and post-APHV children. CONCLUSIONS: Among prepubescent children, the influence of biological maturity on gender differences in PA may be a function of how maturity status is determined. The most physically active prepubescent children were those who were on time according to APHV.