The aim of the present study was to examine the associations of objectively measured physical activity (PA) with several markers of fatness and fitness in a relatively large sample of adolescents with Down syndrome (DS). This study comprised a total of 100 adolescents with DS (37 females) aged 11–20 years-old, and a sex-matched sample of 100 adolescents without disabilities, participating in the UP&DOWN study. The ALPHA health-related fitness test battery for adolescents was used to assess fatness and fitness. PA was measured by accelerometry. Adolescents with DS had higher fatness and significantly lower fitness levels in all variables measured than adolescents without DS (all p <0.05). Moderate-to-large effects were observed in fatness variables (d =0.65–1.42), but particularly large values were found in fitness variables (d =2.05–2.43). In addition, PA levels was not associated with fatness variables, whereas total PA and vigorous PA were associated with all fitness variables (p <0.05), and moderate-vigorous PA (MVPA) was associated with muscular fitness (p <0.05), after adjusting for potential confounders. Further analysis revealed that there were differences in fitness by tertiles of vigorous PA between the lowest and the highest groups in all fitness variables (all p <0.05). However, no significant differences were found in fitness by tertiles of MVPA according with PA guidelines (≥60min in MVPA). Our findings indicate that PA levels are not associated with fatness variables, whereas high PA levels, in particular vigorous PA, are positively associated with high fitness in adolescents with DS.