LI-F type peptides are a family of cyclic lipodepsipeptide antibiotics isolated from Paenibacillus polymyxa and display potent activities against positive bacteria including methicillin-resistant S. aureus (MRSA). In this study, we investigated the mechanism of action of LI-F type peptide AMP-jsa9 against a MRSA (S. aureus CICC10790), which is resistant to ciprofloxacin, gentamicin, kanamycin, chloramphenicol, methicillin, and tetracycline. It was found that AMP-jsa9 mainly targets the cell membrane of MRSA and is able to inhibit biofilm formation through killing planktonic bacteria cells. Moreover, AMP-jsa9 can bind to DNA in vitro, which represents another pathway for the action on MRSA. Furthermore, in vivo treatment of scalded mice with AMP-jsa9 resulted in inhibiting MRSA infections and healing of the scalded wound. In addition, it was demonstrated that AMP-jsa9 can effectively inhibit MRSA infections in scalded murine epidermis and that inflammatory cytokines including IL-8, IL-6, tumor necrosis factor alpha (TNF-alpha), and monocyte chemotactic factor-1 (MCP-1) were reduced; moreover, both protein and gene expression levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (e-NOS) were enhanced, which promote neovascularization and proliferation of new granulation tissue.