Most Canadian children are not meeting the recommended 60 min of moderate-to-vigorous physical activity per day. Research suggests that children’s perceptions of their environment have an influence on their physical activity behaviours, but there is a lack of generalizability among previous work. The purpose of this study was to assess the mediating effect of children’s perceptions of barriers to physical activity on the relationship between their environments and their level of moderate-to-vigorous physical activity (measured with accelerometers). Structural equation modelling stratified by gender was used to assess the research objective in a sample of 546 participants aged 8–14 years old from Northwestern and Southwestern Ontario, Canada. In both models stratified by gender, perceptions of barriers did not significantly mediate the relationship between urbanicity and physical activity. Independent of all other factors, there was no significant relationship between urbanicity and physical activity in girls, but there was in boys. These results offer insight into potential processes by which perceptions impact physical activity and provide initial information to further our understanding of the behavioural aspects of physical activity through multiple levels of analysis. Researchers must continue to improve efforts for quantifying the experience of children’s daily activity contexts.