Whole blood donors, especially frequently donating donors, have a risk of iron deficiency and low hemoglobin (Hb) levels, which may affect their health and eligibility to donate. Lifestyle behaviors, such as dietary iron intake and physical activity, may influence iron stores and thereby Hb levels. We aimed to investigate whether dietary iron intake and questionnaire-based moderate-to-vigorous physical activity (MVPA) were associated with Hb levels, and whether ferritin levels mediated these associations. In Donor InSight-III, a Dutch cohort study of blood and plasma donors, data on heme and non-heme iron intake (mg/day), MVPA (10 minutes/day), Hb levels (mmol/L) and ferritin levels (mg/L) were available in 2,323 donors (1,074 male). Donors with higher heme iron intakes [regression coefficients (β) in men and women: 0.160 and 0.065 mmol/L higher Hb per 1 mg of heme iron, respectively] and lower nonheme iron intakes (β: -0.014 and -0.017, respectively) had higher Hb levels, adjusted for relevant confounders. Ferritin levels mediated these associations [indirect effect (95% confidence interval) in men and women, respectively: 0.074 (0.045; 0.111) and 0.061 (0.030; 0.096) for heme and -0.003 (-0.008;0.001) and -0.008 (-0.013;-0.003) for non-heme]. MVPA was negatively associated with Hb levels in men only (β: -0.005), but not mediated by ferritin levels. In conclusion, higher heme and lower non-heme iron intake were associated with higher Hb levels in donors, via higher ferritin levels. This indicates that donors with high heme iron intake may be more capable of maintaining iron stores to recover Hb levels after blood donation.