Growing data suggest that antibiotic exposure is associated with a long-lasting alteration in gut microbiota, and may be related to subsequent cardiovascular disease (CVD). We investigated associations of life-stage and duration of antibiotic exposure during adulthood with subsequent CVD events. This study included 36 429 women initially free of CVD and cancer from the Nurses' Health Study. We estimated hazard ratios (HRs) for CVD (a composite endpoint of coronary heart disease or stroke) according to duration of antibiotic use in young (age 20-39), middle (age 40-59), and late (age 60 and older) adulthood. During an average of 7.6 years of follow-up, 1056 participants developed CVD. Women with long-term use of antibiotics (for ≥2 months) in late adulthood had a significantly increased risk of CVD (HR 1.32, 95% confidence interval 1.03-1.70) after adjustment for covariates (such as demographic factors, diet and lifestyle, reasons for antibiotic use, overweight or obesity, disease status, and other medication use), as compared to women who did not use antibiotics in this life-stage. Longer duration of antibiotic use in middle adulthood was also related to higher risk of CVD (P trend = 0.003) after controlling for these covariates. There was no significant relationship between the use in young adulthood and the risk of CVD. In this study which examined the antibiotic use in different life-stages, longer duration of exposure to antibiotics in the middle and older adulthood was related to an increased risk of future CVD events among elderly women at usual risk.