BACKGROUND: B-vitamins are essential for one-carbon metabolism and have been linked to colorectal cancer. Although associations with folate have frequently been studied, studies on other plasma vitamins B2, B6, and B12 and colorectal cancer are scarce or inconclusive. METHODS: We carried out a nested case-control study within the European Prospective Investigation into Cancer and Nutrition, including 1,365 incident colorectal cancer cases and 2,319 controls matched for study center, age, and sex. We measured the sum of B2 species riboflavin and flavin mononucleotide, and the sum of B6 species pyridoxal 5'-phosphate, pyridoxal, and 4-pyridoxic acid as indicators for vitamin B2 and B6 status, as well as vitamin B12 in plasma samples collected at baseline. In addition, we determined eight polymorphisms related to one-carbon metabolism. Relative risks for colorectal cancer were estimated using conditional logistic regression, adjusted for smoking, education, physical activity, body mass index, alcohol consumption, and intakes of fiber and red and processed meat. RESULTS: The relative risks comparing highest to lowest quintile were 0.71 [95% confidence interval (95% CI), 0.56-0.91; P(trend) = 0.02] for vitamin B2, 0.68 (95% CI, 0.53-0.87; P(trend) <0.001) for vitamin B6, and 1.02 (95% CI, 0.80-1.29; P(trend) = 0.19) for vitamin B12. The associations for vitamin B6 were stronger in males who consumed ≥30 g alcohol/day. The polymorphisms were not associated with colorectal cancer. CONCLUSIONS: Higher plasma concentrations of vitamins B2 and B6 are associated with a lower colorectal cancer risk. IMPACT: This European population-based study is the first to indicate that vitamin B2 is inversely associated with colorectal cancer, and is in agreement with previously suggested inverse associations of vitamin B6 with colorectal cancer.