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So novel antimicrobial agents were urgently needed to defend the resistant bacteria. Jelleine\u2010I was a small antimicrobial peptide (AMP) with eight amino acids in its sequence. It was believed to be an ideal template for developing antimicrobial agents. In the present study, the possible action mode against both gram\u2010negative bacteria and gram\u2010positive bacteria and in vivo antimicrobial activity was explored. Our results showed that Jelleine\u2010I exhibits its antimicrobial activity mainly by disrupting the integrity of the cell membrane, which would not be affected by the conventional resistant mechanism. It also aims at some intracellular targets such as genomic DNA to inhibit the growth of microbes. In addition, the result of in vivo antimicrobial activity experiment showed that Jelleine\u2010I performed a good therapeutic effect toward the mice with <jats:italic>Escherichia coli<\/jats:italic> infected peritonitis. 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