To examine the associations between dietary fat intake and ischemic stroke among postmenopausal women. We conducted a prospective cohort study of 87,025 generally healthy postmenopausal women (age, 50-79 years) enrolled in the Women's Health Initiative Observational Study. Repeated and validated dietary assessments were done using a self-administered food frequency questionnaire. We used Cox proportional hazards models to estimate hazard ratios (HRs) of ischemic stroke based on quintiles of the cumulative average of fat intake. We documented 1,049 incident cases of ischemic stroke over 663,041 person-years of follow-up. Women in the highest quintile of trans fat intake had a significantly higher incidence of ischemic stroke (HR, 1.39; 95% confidence interval [CI], 1.08-1.79; p-trend = 0.048) compared with women in the lowest quintile, while controlling for multiple covariates. The observed association was modified by aspirin use (p-interaction = 0.02). The HR was 1.66 (95% CI, 1.21-2.36; p-trend < 0.01) among baseline non-aspirin users (n = 67,288) and 0.95 (95% CI, 0.60-1.48; p-trend = 0.43) among aspirin users (n = 19,736). No significant associations were found between intakes of saturated, monounsaturated, or polyunsaturated fat and ischemic stroke or any ischemic stroke subtypes. In this large cohort of postmenopausal women, higher intake of trans fat was associated with incident ischemic stroke independent of major lifestyle/dietary factors. Aspirin use may attenuate the potential adverse effect of trans fat intake on ischemic stroke.