Serum 25-Hydroxyvitamin D (25(OH)D) has been demonstrated to be associated with risk of colorectal cancer (CRC). However, it remains unclear whether this association was modified by vitamin D-related polymorphisms. We evaluated association of serum 25(OH)D concentrations with CRC risk among 403,170 participants from UK Biobank Project. Two variants of vitamin D binding protein (VDBP), rs4588 and rs7041, were included to estimate the binding affinity of 25(OH)D to VDBP, and three variants of vitamin D receptor (VDR), rs11568820, rs2228570, and rs1544410, which may influence VDR activity, were also investigated. Multivariable Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). During 4,957,677 person-years of follow-up, 5053 incident CRC cases were documented. Higher serum 25(OH)D concentrations were significantly associated with lower CRC incidence in a dose-response manner, with HR (95% CIs) being 0.94 (0.91-0.97) per 1 standard deviation (SD) increment of serum 25(OH)D level (Ptrend < 0.001). When separated by anatomic site, we observed a significant association between higher 25(OH)D and lower incidence of colon cancer (Ptrend < 0.001), but not rectal cancer (Ptrend = 0.880). The inverse associations between 25(OH)D level and CRC risk were demonstrated in almost all individuals carrying different GC or VDR genotypes, except for those with rs1544410 TT genotypes or rs4588 TT genotypes. There was no significant interaction between any single variant, or haplotypes of GC or VDR, and 25(OH)D level. Our findings suggest the potential benefits of maintaining adequate vitamin D for CRC prevention, particularly for tumors from colon. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.