Folate plays an important role in DNA methylation, and aberrant methylation of the estrogen receptor (ER) gene may be related to the loss of ER gene expression in breast tumors. Thus, deficient folate status has been hypothesized to be associated primarily with ER gene-negative breast tumors, but data relating folate intake to breast cancer risk according to ER status are sparse. We conducted a prospective cohort analysis of folate intake among 88,744 women in the Nurses' Health Study who completed a food frequency questionnaire in 1980 and every 2 to 4 years thereafter. During 20 years of follow-up, 2,812 ER+ and 985 ER- invasive breast cancer cases were documented. Higher total folate intake was significantly associated with lower risk of developing ER- but not ER+ breast cancer; the multivariable relative risks (RR) and 95% confidence intervals (95% CI) comparing the highest to the lowest quintile were 0.81 (0.66-0.99) for ER- tumors and 1.00 (0.89-1.14) for ER+ tumors. The inverse association between total folate intake and ER- breast cancer was mainly present among women consuming at least 15 g/d of alcohol (multivariable RR, 0.46; 95% CI,=0.25-0.86; top versus bottom quintile). These findings support the hypothesis that higher folate intake reduces the risk of developing ER- breast cancer. Ensuring adequate folate intake seems particularly important for women at higher risk of breast cancer because of alcohol consumption.