BACKGROUND The adequacy of communication and knowledge of donors and physicians regarding iron needs and the relationship between hemoglobin (Hb) and iron stores require evaluation to address donor iron deficiency. METHODS A prospective cohort study was performed on 550 successful donors and 50 donors deferred for low Hb (<125 g/L on repeat fingerstick). Donors participated in an on-clinic interview and had serum ferritin measured. They were mailed their results and recontacted regarding follow-up. RESULTS Most donors are unaware of possible health impacts of donation and do not discuss donation with their physician. In successful donors, mean ferritin levels were 37 and 131 μg/L in first-time and reactivated (no donation for 2 years) females and males and 19 and 29 μg/L in frequent repeat females and males, respectively (p < 0.0001), with infrequent donors having intermediate results. Mean ferritin was 12 μg/L in donors deferred for low Hb. Twenty of 22 donors failing initial Hb testing and passing on repeat testing had ferritin below 25 μg/L. On follow-up, 63 of 164 donors (38%) with low ferritin were taking iron supplements 2 months postdonation. CONCLUSIONS Iron deficiency is frequent, particularly in female donors and frequent donors. A fail on initial Hb testing followed by a pass on repeat testing is likely to be due to iron deficiency and borderline anemia. Donors and physicians need to be more aware of iron needs associated with blood donation and appropriate treatment for low iron stores.