Abstract Background Several studies have determined that dietary intake of B vitamins may be associated with breast cancer risk as a result of interactions between 5,10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) in the one-carbon metabolism pathway. However, the association between B vitamin intake and breast cancer risk in Brazilian women in particular has not yet been investigated. Methods A case-control study was conducted in São Paulo, Brazil, with 458 age-matched pairs of Brazilian women. Energy-adjusted intakes of folate, vitamin B₆, and vitamin B₁₂ were derived from a validated Food Frequency Questionnaire (FFQ). Genotyping was completed for MTHFR A1298C and C677T, and MTR A2756G polymorphisms. A logistical regression model was used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs). Results Neither dietary intake of folate, vitamin B₆, or vitamin B₁₂ nor MTHFR polymorphisms were independently associated with breast cancer risk. Analysis stratified by menopausal status showed a significant association between placement in the highest tertile of folate intake and risk of breast cancer in premenopausal women (OR = 2.17, 95% CI: 1.23–3.83; P _trend = 0.010). The MTR 2756GG genotype was associated with a higher risk of breast cancer than the 2756AA genotype (OR = 1.99, 95% CI = 1.01–3.92; P _trend = 0.801), and statistically significant interactions with regard to risk were observed between the MTHFR A1298C polymorphism and folate (P = 0.024) or vitamin B₆ (P = 0.043), and between the MTHFR C677T polymorphism and folate (P = 0.043) or vitamin B₁₂ (P = 0.022). Conclusion MTHFR polymorphisms and dietary intake of folate, vitamin B₆, and vitamin B₁₂ had no overall association with breast cancer risk. However, increased risk was observed in total women with the MTR 2756GG genotype and in premenopausal women with high folate intake. These findings, as well as significant interactions between MTHFR polymorphisms and B vitamins, warrant further investigation.