B vitamins are involved in 1-carbon metabolism, which is necessary for DNA replication, DNA repair, and regulation of gene expression. Recent studies suggest inverse associations between folate and vitamin B6 intakes and colorectal cancer risk but associations with other B vitamins and methionine have not been widely studied. After following 14,645 men and 22,467 women for 15 yr on average, we ascertained 910 incident colorectal cancers. Dietary intakes were estimated using a 121-item food frequency questionnaire. Hazard ratios (HRs) and 95% confidence intervals were estimated using Cox regression. We found some evidence of a U-shaped relationship between colon cancer risk and vitamin B6 and an inverse U-shaped relationship between rectal cancer risk and B12 (test for the quadratic trend, P = 0.005 and P = 0.0005 respectively). For colon cancer, we observed a reduced risk associated with low methionine/high folate, HR = 0.63 (0.49, 0.80) and an increased risk associated with high methionine/high folate, HR = 1.36 (1.06, 1.74) (P interaction < 0.0001). Our study suggests a U-shaped association between colon cancer risk and vitamin B6 intake and an inverse U-shaped association between rectal cancer risk and vitamin B12. Adequate folate intake might protect against colon cancer risk in those with low methionine intake.