It is not known whether genetic variation in the vitamin D binding protein (DBP) influences 25-hydroxyvitamin D levels [25(OH)D] after vitamin D supplementation. We aimed to investigate the changes of total 25(OH)D, 25(OH)D₃ and 25(OH)D₂ in a Thai cohort, according to type of vitamin D supplement (vitamin D₃ or D₂) and DBP genotype, after receiving vitamin D₃ or D₂ for 3 months. Thirty-nine healthy subjects completed the study. All subjects received 400 IU of either vitamin D₃ or D₂, plus a calcium supplement, every day for 3 months. Total serum 25(OH)D, 25(OH)D₃ and 25(OH)D₂ were measured by LC-MS/MS. Individual genotyping of rs4588 in the DBP gene was performed using real-time PCR. Vitamin D₃ supplementation of 400 IU/d increased 25(OH)D₃ significantly (+16.2 ± 4.2 nmol/L, p <0.001). Vitamin D₂ (400 IU/d) caused increased 25(OH)D₂ levels (+22.0 ± 2.11 nmol/L, p <0.001), together with a decrease of 25(OH)D₃ (-14.2 ± 2.0 nmol/L, p <0.001). At 3 month, subjects in vitamin D3 group tended to have higher total 25(OH)D levels than those in vitamin D₂ (67.8 ± 3.9 vs. 61.0 ± 3.0 nmol/L; p = 0.08). Subjects were then classified into two subgroups: homozygous for the DBP rs4588 C allele (CC), and the rest (CA or AA). With D₃ supplementation, subjects with CA or AA alleles had significantly less increase in 25(OH)D₃ and total 25(OH)D when compared with those with the CC allele. However, no difference was found when the supplement was vitamin D₂. Genetic variation in DBP (rs4588 SNP) influences responsiveness to vitamin D₃ but not vitamin D₂.