The studies investigated the association between serum 25-hydroxyvitamin D (25(OH)D) concentrations and cardiorespiratory fitness (CRF) are few, and the results are controversial. We aim to evaluate the association of serum 25(OH)D concentrations with CRF in adults, and assess whether the associations vary with ageing.The study included 78 middle-aged (30-64 years) and 83 elderly (65-79 years) Japanese men. Hand grip strength and leg extension power were measured using hand grip and leg dynamometers, respectively. CRF was measured via a maximal graded exercise test and quantified as the peak oxygen uptake (VO2peak). Fasting serum 25(OH)D concentrations were determined by enzyme-linked immunosorbant assay.Serum 25(OH)D concentrations were positively related to hand grip strength (r=0.331, p<0.001), leg extension power (r=0.353, p<0.001), CRF (r=0.285, p<0.001) in all adults after adjustment for age. Significant interaction between 25(OH)D and age on CRF was observed (p<0.05). Age-related reduction of CRF was prevented in high 25(OH)D group (p<0.05). In the elderly, subjects in high 25(OH)D group had higher CRF compared with those in low group (p<0.05).We found that serum 25(OH)D concentrations significantly related with leg extension power, hand grip strength and CRF after adjustment for age. The relationship of vitamin D and CRF was affected by ageing. Higher serum 25(OH)D concentrations are a key predictor for CRF in the elderly, but not in middle-aged men.