BACKGROUND: Vitamin D concentrations are linked to body composition indices, particularly body fat mass. Relationships between hypovitaminosis D and obesity, described by both BMI and waist circumference, have been mentioned. We have investigated the effect of a 12-week vitamin D3 supplementation on anthropometric indices in healthy overweight and obese women. METHODS: In a double-blind, randomized, placebo-controlled, parallel-group trial, seventy-seven participants (age 38 ± 8.1 years, BMI 29.8 ± 4.1 kg/m²) were randomly allocated into two groups: vitamin D (25 μg per day as cholecalciferol) and placebo (25 μg per day as lactose) for 12 weeks. Body weight, height, waist, hip, fat mass, 25(OH) D, iPTH, and dietary intakes were measured before and after the intervention. RESULTS: Serum 25(OH)D significantly increased in the vitamin D group compared to the placebo group (38.2 ± 32.7 nmol/L vs. 4.6 ± 14.8 nmol/L; P<0.001) and serum iPTH concentrations were decreased by vitamin D3 supplementation (-0.26 ± 0.57 pmol/L vs. 0.27 ± 0.56 pmol/L; P<0.001). Supplementation with vitamin D3 caused a statistically significant decrease in body fat mass in the vitamin D group compared to the placebo group (-2.7 ± 2.1 kg vs. -0.47 ± 2.1 kg; P<0.001). However, body weight and waist circumference did not change significantly in both groups. A significant reverse correlation between changes in serum 25(OH) D concentrations and body fat mass was observed (r = -0.319, P = 0.005). CONCLUSION: Among healthy overweight and obese women, increasing 25(OH) D concentrations by vitamin D3 supplementation led to body fat mass reduction.