To establish an efficient expression system for a fusion protein of glutathione S-transferase and cecropin B (GST-CB) and to clarify the antibacterial mechanism of CB. The optimal incubation time and methanol concentration for induced expression of CB were 36 h and 1 % w/v, respectively. The yield of GST-CB was 2.2 g/l. The minimum inhibitory concentrations of GST-CB towards Staphylococcus aureus subsp. saprophyticus (ATCC 15305) and Escherichia coli strain CFT073 were 250 and 125 mu g/ml, respectively. Notably, mutations of proline 24 (P24) in CB produced a polypeptide without antimicrobial activity. The fusion protein GST-CB, which has a broad spectrum antimicrobial activity, can be abundantly expressed in Pichia pastoris GS115, and P24 may be an important amino acid for the antimicrobial activity of GST-CB.