The misuse of antibiotics has led our age to a dangerous edge, as antibiotic-resistant pathogens appear to evolve more quickly than antibiotics are invented. Thus, new agents to treat bacterial infection are badly needed. Cationic host defense peptides are on the first line of a host defense system and are thought to be good candidates for treating bacterial infection. Here, a novel cationic host defense peptide, mucroporin, was cloned and characterized from the venom of Lychas mucronatus. The MIC for Staphylococcus aureus was 25 microg/ml, including antibiotic-resistant pathogens. Based on the molecular template of mucroporin, mucroporin-M1 was designed by amino acid substitution. The MIC for S. aureus was 5 microg/ml, including the antibiotic-resistant pathogens methicillin-resistant S. aureus, methicillin-resistant coagulase-negative Staphylococcus, penicillin-resistant S. aureus, and penicillin-resistant S. epidermidis. Moreover, mucroporin-M1 also inhibited gram-negative bacteria. The modes of action of mucroporin and mucroporin-M1 were both rapid killing by disrupting the cell membrane of bacteria, and the number of surviving bacteria was reduced by about 4 to 5 orders of magnitude immediately after peptide delivery. These results showed that mucroporin could be considered a potential anti-infective drug, especially for treating antibiotic-resistant pathogens.