Accumulating evidence from epidemiologic studies suggests that risk of breast cancer is reduced in relation to increased consumption of folate and related B vitamins. We investigated independent and joint effects of B vitamin intake as well as two polymorphisms of a key one-carbon metabolizing gene [i.e., methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C] on breast cancer risk. The study uses the resources of a population-based case-control study, which includes 1,481 cases and 1,518 controls. Significant inverse associations between B vitamin intake and breast cancer risk were observed among non-supplement users. The greatest reduction in breast cancer risk was observed among non-supplement users in the highest quintile of dietary folate intake [odds ratio (OR), 0.61; 95% confidence interval (95% CI), 0.41-0.93] as compared with non-supplement users in the lowest quintile of dietary folate intake (high-risk individuals). The MTHFR 677T variant allele was associated with increased risk of breast cancer (P, trend = 0.03) with a multivariate-adjusted OR of 1.37 (95% CI, 1.06-1.78) for the 677TT genotype. The 1298C variant allele was inversely associated with breast cancer risk (P, trend = 0.03), and was likely due to the linkage of this allele to the low-risk allele of 677C. The MTHFR-breast cancer associations were more prominent among women who did not use multivitamin supplements. Compared with 677CC individuals with high folate intake, elevation of breast cancer risk was most pronounced among 677TT women who consumed the lowest levels of dietary folate (OR, 1.83; 95% CI, 1.13-2.96) or total folate intake (OR, 1.71; 95% CI, 1.08-2.71). From a public heath perspective, it is important to identify risk factors, such as low B vitamin consumption, that may guide an effective prevention strategy against the disease.