Objectives: To assess the association between obesity (measured by Body Mass Index (BMI) and fat percentage) and serum 25(OH)D levels in older persons. Design: Cross-sectional analysis of data from ‘the B-PROOF study’ (B-vitamins for the Prevention Of Osteoporotic Fractures). Participants: 2842 participants aged 65 years and older. Measurements: BMI and fat percentage, measured by Dual Energy X-ray, and serum 25(OH) D levels. Results: Mean age was 74 years (6.5 SD), with 50% women. Mean serum 25(OH)D levels were 55.8 nmol/L (25 SD). BMI and total body fat percentage were significant inversely associated with serum 25(OH)D levels after adjustment for confouders (β−0.93; 95%CI [−1.15; −0.71], p<0.001 and β−0.84; 95%CI [−1.04; −0.64], p<0.001). This association was most prominent in individuals with a BMI in the ‘overweight’ and ‘obesity’ range (β−1.25 and −0.96 respectively) and fat percentage in the last two upper quartiles (β−1.86 and −1.37 respectively). Conclusion: In this study, higher BMI and higher body fat percentage were significantly associated with lower serum 25(OH)D levels in older persons. This association was particularly present in individuals with overweight, and higher fat percentages, suggesting that these persons are at increased risk of vitamin D insufficiency.