Abstract Background: Vitamin B6 has been hypothesized to play a role in colorectal carcinogenesis through its one-carbon metabolism related DNA synthesis and methylation. However, observational studies examining associations between vitamin B6 intake and colorectal cancer risk have been inconclusive and no studies have investigated the optimal timing of vitamin B6 intake for colorectal cancer prevention. Method: We prospectively followed 86440 women in the Nurses’ Health Study and 45255 men in the Health Professionals Follow-up Study for up to 28 years. We assessed vitamin B6 intakes every 4 years using validated food frequency questionnaires. We evaluated whether higher vitamin B6 intake in the remote past (i.e., 12-16 years) is more strongly associated with a lower risk of colorectal cancer than intake in the recent past (i.e., 0-4 years, 4-8 years). Cox proportional hazards regression models were used to estimate multivariable relative risks (MV RRs, 95%CIs). Results: The mean intakes of vitamin B6 were above the Recommended Daily Allowance (RDA) levels in each cohort. Total vitamin B6 intake was associated with plasma pyridoxal 5′-phosphate (PLP, the active form of vitamin B6) levels. Comparing top with bottom quintile categories of total vitamin B6 intakes, the mean plasma PLP levels were 98.3 pmol/mL and 38.9 pmol/mL in women and were 183.2 pmol/mL and 66.0 pmol/mL in men. Total vitamin B6 intake was significantly associated with an approximately 20-30% lower risk of colorectal cancer in age-adjusted results. However, significant inverse associations became attenuated and non-significant after further adjustment for body mass index, physical activity, family history of colorectal cancer, aspirin use, screening, and other dietary factors including intakes of vitamin D and folate. Compared highest to lowest quintile of cumulative intake of total vitamin B6, the MV RRs (95%CIs) for colorectal cancer were 0.98 (0.80, 1.22; P trend = 0.79) in women and 0.98 (0.76, 1.26; P trend = 0.60) in men. For the same comparison, the MV RRs were 0.92 (0.73, 1.16) for total vitamin B6 intake 0-4 year before diagnosis, 0.99 (0.78, 1.26) for intakes 4-8 year before diagnosis, 0.93 (0.71, 1.21) for intakes 8-12 year before diagnosis, and 0.93 (0.69, 1.26) for intakes 12-16 years before diagnosis. The corresponding MV RRs for men were 0.85 (0.63, 1.16), 0.98 (0.70, 1.37), 0.90 (0.63, 1.28), and 1.19 (0.78, 1.83). Additionally, results did not differ by cancer sub-site, sources of vitamin B6 (food or supplement), intakes of alcohol and folate, riboflavin, B12, methionine, body mass index, or family history of colorectal cancer. Conclusion: Although a small effect of vitamin B6 on colorectal cancer risk cannot be excluded, our prospective data do not support a strong role of vitamin B6 intake in adulthood in colorectal carcinogenesis among middle-aged U.S. health professionals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 628. doi:1538-7445.AM2012-628