CYP24A1 and CYP27B1 are critical genes determining 1α,25(OH)D concentration and impacting on carcinogenesis. A case-control study including 528 colorectal cancer (CRC) patients and 605 cancer-free controls and a follow-up study with 317 cases were conducted in northeast China. Genotypes were tested by TaqMan Genotyping Assays. Individuals carrying the GG genotype of CYP27B1 G > T (rs10877012) exhibited decreased CRC risk compared with those with the TT genotype (OR (OR) = 0.57, 95% Confidence Interval (CI) = 0.38-0.84). Compared with the TT genotype, a significant association between the CC genotype of CYP27B1 C > T (rs4646536) and a reduced risk of CRC was observed (OR = 0.59, 95% CI = 0.40-0.88). We also observed significant combined effects of the two polymorphisms in CYP27B1 with dietary factors, including the intake of cereals, overnight meal, allium vegetables, pork, canned fruit, and braised fish, on CRC risk. These associations remained significant after Bonferroni correction for multiple comparisons. The Hazard Ration (HR) of patients with the AA genotype (CYP24A1 A > G, rs4809957) was 2.38 (95% CI = 1.30-4.37) when compared with the GG genotype. Thus, our findings suggested that two polymorphisms in CYP27B1 are associated with CRC susceptibility. CYP24A1 A > G (rs4809957) polymorphism may lead to a worse prognosis of CRC.