Hepcidin was first identified as an antimicrobial peptide expressed in the liver. It was later demonstrated that hepcidin is in fact the long sought hormone that regulates iron homeostasis in mammals. Hepcidin is encoded as an 84 amino acid prepropeptide that is successively cleaved to yield prohepcidin and the mature 25 amino acid hepcidin. Both the bioactive 25-aa hepcidin and the 35-aa proregion are secreted by liver hepatocytes. The aim of the present study was to assess the antibacterial activity of the proregion peptide from human hepcidin. Using a chemically synthesized peptide corresponding to the proregion, we show that it is bactericidal against Bacillus megaterium (25µM), and inhibits Bacillus subtilis growth at high concentration (200µM). No synergistic interaction of proregion and Hepc25 against Bacillus megaterium was seen. In a further step, the mode of action of proregion on Bacillus megaterium was studied. It caused a slow accumulation of the vital stain SYTOX in the bacteria, indicating that it did not destroy the microbial membranes through a detergent-like mechanism, even at concentrations (80µM) higher than those required to kill the bacteria. This result suggests that the target of proregion might be an intracellular component. Finally, gel retardation assay showed that the DNA binding ability of the hepcidin proregion was equivalent to that observed for magainin 2, an antimicrobial peptide which exerts its antimicrobial effect by interfering with intracellular nucleic acids. In conclusion, we speculate that the proregion of hepcidin may have bacteriostatic effects, and as such may contribute to the innate immune response.