SummaryA high number of blood donations may cause iron depletion. The pathophysiology behind this process may involve hepcidin, a recently discovered peptide that acts by inhibiting iron absorption and promoting iron retention in reticuloendothelial macrophages. The aim of this study was to determine serum pro‐hepcidin levels and iron metabolism parameters in multi‐time blood donors. The study group consisted of 132 multi‐time male blood donors and 25 healthy male volunteers (nondonors). Complete blood cell count and iron status including serum iron, ferritin, soluble transferrin receptor (sTfR), total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), erythropoietin and pro‐hepcidin (ELISA) were assessed. In blood donors, ferritin level drops markedly in relation to donation frequency (P < 0.001). In contrast, TIBC and UIBC levels increase progressively corresponding to annual donation frequency. Pro‐hepcidin concentration increases significantly with the number of donations per year (P = 0.0290). In blood donors having donated blood with the highest frequency per year, pro‐hepcidin levels were positively correlated with haemoglobin (R = 0.31, P < 0.05) and negatively with sTfR (R = −0.31, P < 0.05). Pro‐hepcidin levels increase in relation to blood donation frequency per year. Longitudinal studies focusing on changes in serum hepcidin levels are required to address the question whether hepcidin may contribute to iron metabolism disturbances in multi‐times blood donors.