Objective: To investigate whether regular blood donation decreases cardiovascular risk. Methods: All 159 934 Dutch whole-blood donors with an active donation career of at least 10 years were categorised into sex-specific donation tertiles based on the number of donations during this 10-year qualification period. Cardiovascular endpoints were based on hospital discharge diagnoses and death certificates from Dutch Hospital Data and Statistics Netherlands and occurring after the 10-year qualification period. Cox regression was used to estimate the age-adjusted and starting year-adjusted hazard rate ratio (HRR). Results: Female high-frequency blood donors had a reduced cardiovascular morbidity (HRR=0.91, 95% CI 0.85 to 0.98) compared with low-frequency blood donors. No effect was observed in men (HRR=1.00, 95% CI 0.95 to 1.05). To rule out a residual healthy donor effect (HDE), additional sensitivity analyses using a 5-year qualification period were conducted. The results supported the absence of a residual HDE. Conclusions: This study showed a protective effect of long-term, high-frequency blood donation against cardiovascular disease. This effect was only observed in women and not in men.