BACKGROUND: Vitamins B2, B6, and B12 are key players in one-carbon metabolism as enzymatic cofactors, and deficiency of these nutrients may influence reproductive outcomes possibly through affecting reproductive hormones. OBJECTIVE: The goal was to investigate associations between dietary intakes of vitamin B2, B6, and B12, and menstrual function among premenopausal women. DESIGN: This was a secondary analysis of a prospective cohort study conducted at the University at Buffalo in 2005-2007. PARTICIPANTS/SETTING: Participants were 259 healthy, regularly-menstruating women (aged 18-44 years) with self-reported menstrual cycles between 21 and 35 days, who were not trying to conceive, and who had not used hormonal contraception in the past three months. MAIN OUTCOME MEASURE(S): Intakes of B-vitamins were assessed via 24-hour dietary recalls four times per menstrual cycle for two cycles. Serum reproductive hormones and plasma homocysteine were measured eight and three times, respectively, per cycle for two cycles. Anovulatory cycles were determined by progesterone concentrations ≤5 ng/mL (15.9 nmol/L) and no observed serum luteinizing hormone peak during the mid or late luteal phase visit. STATISTICAL ANALYSIS: Weighted linear mixed regressions were used to evaluate associations between cycle-averaged B-vitamin intakes and hormones and homocysteine, and generalized linear regressions for associations with anovulation. Models were adjusted for age, race, BMI, physical activity, alternate Mediterranean diet score, intakes of total energy, protein, fiber, and folate, and percentage of energy intake from fat. RESULTS: Higher intakes of vitamin B2 (per 0.1 mg increase in intake) were inversely correlated with estradiol (−0.87%, 95% confidence interval [CI] −1.67, −0.06) and homocysteine levels (−0.61%, 95% CI −1.10, −0.12). Higher vitamin B6 intakes were suggestive of higher follicle-stimulating hormone though the results were not statistically significant (0.63% difference, 95% CI −0.03, 1.29, per 0.1 mg increase in intake; P=0.06). Small increases in testosterone and decreases in homocysteine were found with vitamin B12 intake. No associations were observed between intake of B-vitamins and a risk of sporadic anovulation. CONCLUSION: Higher intakes of vitamin B2 were associated with a small decrease in serum estradiol among healthy, regularly-menstruating women. Higher intakes of B2 and B12 were associated with lower plasma homocysteine concentrations. Overall, vitamins B2, B6, and B12 that are one-carbon nutrients do not appear to influence the ovarian cycle among premenopausal women.