Relationships between gross motor skills and cardiovascular fitness with visuospatial working memory (VSWM) in children are hypothesized to be mediated by underlying functional brain mechanisms. Because there is little experimental evidence to support this mechanism, the present study was designed to investigate the relationships of gross motor skills and cardiovascular fitness with VSWM-related brain activation in 8- to 10-year-old children. Functional magnetic resonance imaging data obtained during a VSWM-task were analyzed for 80 children from grades 3 (47.5%) and 4 of 21 primary schools in the Netherlands (51.3% girls). Gross motor skills (Korper Koordinationstest für Kinder and Bruininks-Oseretsky Test of Motor Proficiency - 2nd Edition) and cardiovascular fitness (20-meter Shuttle Run Test) were assessed. VSWM-related brain activation was found in a network involving the angular gyrus, the superior parietal cortex, and the thalamus; deactivation was found in the inferior and middle temporal gyri. Although behavioral results showed significant relations of gross motor skills and cardiovascular fitness with VSWM performance, gross motor skills and cardiovascular fitness were not related to VSWM-related brain activation. Therefore, we could not confirm the hypothesis that brain activation underlies the relationship of gross motor skills and cardiovascular fitness with VSWM performance. Our results suggest that either the effects of physical activity on cognition do not necessarily go via changes in gross motor skills and/or cardiovascular fitness, or that brain activation patterns as measured with the blood-oxygen-level dependent (BOLD) signal may not be the mechanism underlying the relationships of gross motor skills and cardiovascular fitness with VSWM.