B vitamins, such as folate, vitamin B6, and vitamin B12, play an important role as coenzymes in one-carbon metabolism and may affect colorectal cancer risk. We aimed to comprehensively investigate the relationships of plasma folate, pyridoxal-5'-phosphate (PLP, the active form of vitamin B6), vitamin B12, methylmalonic acid, homocysteine, and cysteine with colorectal cancer risk, accounting for suspected modifiers (alcohol intake, MTHFR C677T genotype, and plasma C-reactive protein) and potential confounders. We conducted a case-control study nested within the Multiethnic Cohort study and analyzed prospectively collected blood samples from 224 incident colorectal cancer cases and 411 controls matched on age, sex, race/ethnicity, study site, date/time of blood draw, and hours of fasting. We found an inverse association between plasma PLP levels and colorectal cancer, with odds ratios (95% confidence intervals) for increasing quartiles of 1.00, 0.84 (0.51-1.40), 0.62 (0.37-1.03), and 0.49 (0.29-0.83), with P trend = 0.009. This association was not explained by an association with plasma folate, seemed to be stronger at low levels of alcohol intake and among individuals with the MTHFR 677TT genotype, and was independent of plasma C-reactive protein levels. An inverse association with plasma folate was also observed among individuals with a low level of alcohol intake. These data suggest an independent role for vitamin B6 in reducing colorectal cancer risk.