Hepcidin is a liver_expressed, small cysteine rich peptide that acts as a regulator of systemic iron homeostasis. In this work, according to the partiality codon of Pichia pastoris, a DNA fragment containing the coding sequence of hepcidin was designed and synthesized, especially a Kex2 signal cleavage site was fused in 5′end of the antibacterial peptide genes. Then the modified hepcidin gene was inserted into the Pichia pastoris expression vector plasmid pPICZα_A. After electroporation of the resulting vector, pPICZα_A_Hepc, into the yeast host strain GS115, transformants with high copy inserts were selected by 1500mg/L Zeocin selection. Under the control of the promoter AOX1 (alcohol oxidase 1), recombinant hepcidin secreted from P. pastoris had a molecular weight of 2.7kD. After optimization of the flask_shaking culture fermentation, the yield of hepcidin reached 100mg/L in the clarified broth. Through antibacterial assay, the recombinant hepcidin displayed obvious antibacterial activity against Bacillus subtilis. But it could not distinctly inhibit the growth of E. coli BL21(DE3).