Abstract Context Single-nucleotide polymorphisms (SNPs) in genes related to vitamin D metabolism have been associated with serum 25-hydroxyvitamin D 25(OH)D concentration, but these relationships have not been examined following antenatal cholecalciferol supplementation. Objective To determine whether SNPs in DHCR7, CYP2R1, CYP24A1, and GC are associated with the response to gestational cholecalciferol supplementation. Design Within-randomization group analysis of the Maternal Vitamin D Osteoporosis Study trial of antenatal cholecalciferol supplementation. Setting Hospital antenatal clinics. Participants In total, 682 women of white ethnicity (351 placebo, 331 cholecalciferol) were included. SNPs at rs12785878 (DHCR7), rs10741657 (CYP2R1), rs6013897 (CYP24A1), and rs2282679 (GC) were genotyped. Interventions 1000 IU/d cholecalciferol from 14 weeks of gestation until delivery. Main Outcome Measure 25(OH)D at randomization and 34 weeks of gestation were measured in a single batch (Liaison; Diasorin, Dartford, UK). Associations between 25(OH)D and the SNPs were assessed by linear regression using an additive model β represents the change in 25(OH)D per additional common allele. Results Only rs12785878 (DHCR7) was associated with baseline 25(OH)D β = 3.1 nmol/L; 95% confidence interval (CI), 1.0 to 5.2 nmol/L; P < 0.004. In contrast, rs10741657 (CYP2R1) (β = −5.2 nmol/L; 95% CI, −8.2 to −2.2 nmol/L; P = 0.001) and rs2282679 (GC) (β = 4.2 nmol/L; 95% CI, 0.9 to 7.5 nmol/L; P = 0.01) were associated with achieved 25(OH)D status following supplementation, whereas rs12785878 and rs6013897 (CYP24A1) were not. Conclusions Genetic variation in DHCR7, which encodes 7-dehyrocholesterol reductase in the epidermal vitamin D biosynthesis pathway, appears to modify baseline 25(OH)D. In contrast, the response to antenatal cholecalciferol supplementation was associated with SNPs in CYP2R1, which may alter 25-hydroxylase activity, and GC, which may affect vitamin D binding protein synthesis or metabolite affinity. Associations between SNPs in the vitamin D metabolism pathway and 25(OH)D were assessed before and after antenatal vitamin D supplementation. The associations differed at the two time points.