Previous studies suggest that circulating 25(OH)D may favorably influence cardiorespiratory fitness and fat oxidation. However, these relationships have not been examined in older adult women of different ethnic groups. The objectives of this study were to determine whether serum 25(OH)D is related to cardiovascular fitness (VO2max) in sedentary women ages ≥60 years and to determine whether these associations differ between African Americans (AA) and European Americans (EA). A secondary aim was to determine whether serum 25(OH)D is correlated with respiratory quotient (RQ) during submaximal exercise. This cross-sectional analysis included 67 AA and EA women ages 60-74 years. VO2max was measured by a modified Bruce graded treadmill protocol, and measurements were adjusted for percent fat and lean body mass assessed by air displacement plethysmography. Indirect calorimetry was used to measure RQ at rest and during four submaximal exercise tests. Fasting blood samples were obtained to quantify serum 25(OH)D. Serum 25(OH)D was associated with VO2max (ml/kg LBM/min) independent of percent body fat (r = 0.316, p = 0.010). However, subgroup analysis revealed that this relationship was specific to AA (r = 0.727, p = 0.005 for AA; r = 0.064, p = 0.643 for EA). In all subjects combined, 25(OH)D was inversely correlated (p < 0.01) with all measures of submaximal RQ. Higher serum 25(OH)D was associated with greater cardiorespiratory fitness in older adult AA women. Among both AA and EA, inverse associations between serum 25(OH)D and RQ suggest that women with higher levels of circulating vitamin D also demonstrated greater fat oxidation during submaximal exercise.