Low cardiorespiratory fitness and low serum 25-hydroxy vitamin D (25[OH]D) levels are associated with increased cardiovascular and all-cause mortality, but whether low 25(OH)D is independently associated with cardiorespiratory fitness in healthy adults is not known. We examined 25(OH)D levels and fitness in 200 healthy adults participating in a double-blind clinical trial investigating statins and muscle performance (STOMP study). Maximal aerobic exercise capacity (Vo 2max ) was measured using metabolic gas analysis during graded treadmill exercise to exhaustion. 25(OH)D was measured using an enzyme-linked immunosorbent assay. Daily physical activity was assessed using the Paffenbarger Physical Activity Questionnaire. Serum 25(OH)D concentration was positively related to Vo 2max (r = 0.29, p = 0.0001), even after adjusting for relevant predictors (e.g., age, gender, and body mass index). There was also a significant interaction between 25(OH)D level and self-reported hours of moderate to vigorous physical activity (MVPA; p <0.02). With each SD increase in 25(OH)D, Vo 2max increased by 2.6 ml/kg/min (p = 0.0001) when MVPA was low (16 hours/week) and 1.6 ml/kg/min (p <0.0004) when MVPA was moderate (35 hours/week) but only 0.01 ml/kg/min (p = 0.9) when MVPA was high (64 hours/week). In conclusion, serum 25(OH)D levels predict Vo 2max in adults; the effect is greatest in those with low levels of physical activity.