Objective: The influence of vitamin D receptor (VDR) genetic variation on serum 25-hydroxyvitamin D levels [25(OH)D] after vitamin D3 supplementation remains unclear. We aimed to investigate changes of 25(OH)D in a randomized, double-blind, placebo-controlled clinical trial, according to VDR genotype, after provision of vitamin D3 to breast cancer cases for a 2-month period. Methods: Participants were assigned to two treatment arms: placebo (n = 28) and vitamin D3 supplementation (n =28). The supplementation group received 50,000 IU of vitamin D every week for 2 months. Blood samples were collected at baseline and after intervention to measure serum 25(OH)D3. Genotypes were assessed for FokI, BsmI, ApaI, and TaqI polymorphisms. Results: After eight weeks supplementation, the intervention group showed a significant increase in the serum concentration of 25 (OH)D3 (28±2.6 to 39±3.5; p=0.004). Subjects were then classified into twelve subgroups according to different VDR genotypes. Subjects with ff/Ff, TT/Tt, and Bb genotypes had significantly higher increases in serum 25(OH)D compared to those with FF, tt, and BB/bb genotypes post-intervention. Serum vitamin D3 levels with the AA genotype were lower than with aa/ Aa. No differences were found among other subgroups. Conclusion: Vitamin D3 supplementation increases serum 25(OH)D in women with breast cancer. Serum vitamin D3 in TT/Tt, ff/Ff, and Bb carriers was more responsive to vitamin D supplementation than in those with FF/ff and tt genotypes. Other subgroups might gain less from vitamin D3 supplementation.