Despite preclinical evidence supporting anti-cancer effects of cardiac glycosides, epidemiologic studies consistently show elevated breast cancer risk in digoxin users. We studied this association in the Nurses' Health Study cohort to evaluate influences of screening mammography and lifestyle-related risk factors. We followed 90,202 postmenopausal women from 1994 to 2010. Self-reported breast cancers were confirmed by medical record review. We fit Cox regression models to estimate associations between time-varying digoxin use and breast cancer incidence, overall and by tumor ER status, accounting for mammography screening and established breast cancer risk factors. There were 5,004 digoxin users over 1.05 million person-years of observation, among whom 144 breast cancer cases occurred. Digoxin users were more likely to undergo mammographic screening, to be former users of postmenopausal hormones, and to take other medications than never-users; the groups were similar on reproductive history and alcohol consumption. Current digoxin use of >4-year duration was associated with a 45 % increased rate of breast cancer compared with never use (HRadj = 1.45, 95 % CI 1.13-1.86). The association appeared stronger for ER-positive disease (HRadj = 1.46, 95 % CI 1.10-1.95) than for ER-negative disease (HRadj = 1.12, 95 % CI 0.52-2.37). Associations were robust to restriction on regular mammography use and to adjustment for established breast cancer risk factors, including lifestyle-related exposures. The positive association between digoxin use and breast cancer occurrence was not attenuated when lifestyle-related breast cancer risk factors and screening practices were accounted for. Digoxin, a common cardiac drug worldwide, may promote breast carcinogenesis.