Abstract Background In short-term trials, aspirin is associated with gastrointestinal bleeding. However, the effect of dose and duration of aspirin use on risk remains unclear. Methods We conducted a prospective study of 87,680 women enrolled in the Nurses' Health Study in 1990 who provided biennial data on aspirin use. We examined the relative risk (RR) of major gastrointestinal bleeding requiring hospitalization or blood transfusion. Results During a 24-year follow-up, 1537 women reported a major gastrointestinal bleeding. Among women who used aspirin regularly (≥2 standard 325 mg tablets/week), the multivariate RR of gastrointestinal bleeding was 1.43 (95% confidence interval CI, 1.29-1.59) when compared with nonregular users. Compared with women who denied any aspirin use, the multivariate RRs of gastrointestinal bleeding were 1.03 (95% CI, 0.85-1.24) for women who used 0.5 to 1.5 standard aspirin tablets/week, 1.30 (95% CI, 1.07-1.58) for women who used 2 to 5 tablets/week, 1.77 (95% CI, 1.44-2.18) for women who used 6 to 14 tablets/week, and 2.24 (95% CI, 1.66-3.03) for women who used more than 14 tablets/week ( P trend < .001). Similar dose-response relationships were observed among short-term users (≤5 years; P trend <.001) and long-term users (>5 years; P trend <.001). In contrast, after adjustments were made for dose, increasing duration of use did not confer a greater risk of bleeding ( P trend = .28). Conclusion Regular aspirin use is associated with gastrointestinal bleeding. Risk seems more strongly related to dose than duration of aspirin use. Efforts to minimize adverse effects of aspirin therapy should emphasize using the lowest effective dose among both short- and long-term users.