•Vitamin D deficiency is common in the Middle East.•Response to Vitamin D supplementation is genetically influenced.•SNPs in GC gene can predict response to vitamin D supplementation.•Carriers of rs4588/rs7041 in GC had lowest response to vitamin D supplementation. The aim of this study was to determine the influence of vitamin D–binding protein (DBP) gene polymorphisms in vitamin D metabolites before and after vitamin D supplementation. In all, 234 participants (126 women; 108 men) with vitamin D deficiency 25(OH)D <50 nmol/L were given 50 000 IU of vitamin D supplements for 8 wk followed by daily maintenance of 1000 IU for 4 mo. Two single-nucleotide polymorphisms (rs4588 and rs7041) in DBP coding gene were assessed. Baseline 25(OH)D was significantly in higher in participants with homozygous major genotype of rs7041 than other genotypes (P = 0.02). Postsupplementation 25(OH)D was significantly higher in participants with homozygous major genotypes of either rs4588 and rs7041 than other genotypes (P < 0.001). Participants with the minor allele of either rs4588 or rs7041 were 2.9 (1.9–4.5) times and 3.7 (2.1–6.6) times, respectively, more likely to be non-responders (postsupplementation 25 OHD <50 nmol/L) than those homozygous for the major allele at these locations (P < 0.001). Furthermore, participants with homozygous minor and heterozygous genotype of rs7041 were 6.2 and 4.2times more likely to be non-responders than those with the homozygous major genotype (P < 0.001) even after adjustments for age, sex, body mass index, baseline 25(OH)D concentration, and other alleles. Participants with homozygous minor and heterozygous genotypes of rs4588 were 4.1 and 12.4times more likely to be non-responders than those with homozygous major genotypes. These significant risks, however, were lost after adjustment. rs7041 and rs4588 variants of the DBP gene are associated with variations in 25(OH)D levels and efficacy of response to vitamin D supplementation in Saudi Arabian adults.