Cholecalciferol (D3) supplementation results in variable increases in serum 25(OH)D3 levels, however, the influence of genetic polymorphisms on these variable responses is unclear. We measured serum 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and VDBP levels in 50 colorectal cancer (CRC) patients before and during 2,000 IU daily oral D3 supplementation for six months and in 263 archived CRC serum samples. Serum PTH levels and PBMC 24-OHase activity were also measured during D3 supplementation. TagSNPs in CYP2R1, CYP27A1, CYP27B1, CYP24A1, VDR, and GC genes were genotyped in all patients, and the association between these SNPs and serum vitamin D3 metabolites levels before and after D3 supplementation was analyzed. The mean baseline serum 25(OH)D3 level was less than 32 ng/mL in 65 % of the 313 CRC patients. In the 50 patients receiving D3 supplementation, serum levels of 25(OH)D3 increased (p = 0.008), PTH decreased (p = 0.036) and 24,25(OH)2D3, 1,25(OH)2D3, VDBP levels and PBMC 24-OHase activity were unchanged. GC SNP rs222016 was associated with high 25(OH)D3 and 1,25(OH)2D3 levels at baseline while rs4588 and rs2282679 were associated with lower 25(OH)D3 and 1,25(OH)2D3 levels both before and after D3 supplementation. CYP2R1 rs12794714 and rs10500804 SNPs were significantly associated with low 25(OH)D3 levels after supplementation but not with baseline 25(OH)D3. Our results show that D3 supplementation increased 25(OH)D3 levels in all patients. GC rs4588 and rs2283679 SNPs were associated with increased risk of vitamin D3 insufficiency and suboptimal increase in 25(OH)D3 levels after D3 supplementation. Individuals with these genotypes may require higher D3 supplementation doses to achieve vitamin D3 sufficiency.