OBJECTIVE: To investigate the effect of a 9-month physical activity intervention on cardiorespiratory fitness and adiposity among prepubertal children. METHODS: Prepubertal children (8- to 9-year-olds, N = 220, 103 girls) were randomly assigned to a 9-month physical activity intervention or a control group. The intervention provided 70 minutes (5 days/week) of moderate to vigorous physical activity. Maximum oxygen consumption (Vo2max percentile) and dual-energy radiograph absorptiometry measured cardiorespiratory fitness and adiposity, respectively. Intention-to-treat analysis was performed to assess baseline and follow-up cardiorespiratory fitness, percentage fat mass (%FM), percentage central fat mass (%CFM), and estimated visceral adipose tissue (VAT) area. RESULTS: The intervention group increased in cardiorespiratory fitness (5.4th percentile; 95% confidence interval [CI], 1.8 to 8.9) and decreased in %FM (−0.7%; 95% CI, −1.1 to −0.4) and %CFM (−1.3%; 95% CI, −1.9 to −0.7). Reductions in %FM were evident for both nonoverweight (−0.62%; 95% CI, −1.07 to −0.17) and overweight or obese (−0.86%; 95% CI,−1.46 to −0.25) intervention participants. Conversely, the control group displayed no change in cardiorespiratory fitness while exhibiting increases in %FM (0.4%; 95% CI, 0.1 to 0.7), %CFM (0.6; 95% CI, 0.1 to 1.1), and VAT area (3.0 cm2; 95% CI, 1.6 to 4.4). Nonoverweight control participants increased in %FM (0.52%; 95% CI, 0.13 to 0.91), and their overweight and obese counterparts increased in VAT (4.76 cm2; 95% CI, 1.90 to 7.63). CONCLUSIONS: The physical activity intervention improved cardiorespiratory fitness, reduced %FM, and prevented accumulation of %CFM among prepubertal children with varying adiposity levels. These findings provide support for daily physical activity recommendations to prevent excess fat mass accumulation in childhood.