The rapid increase in carbapenem-resistant (CRKP) infections, along with the cross-resistance of CRKP to other antibiotics, has created an urgent need for novel therapeutic agents. Among the potential options for next-generation antibiotics, antimicrobial peptides (AMPs) show great promise. In this study, we aimed to elucidate the mechanisms underlying the antibacterial activity against CRKP of an antibacterial peptide named Cecropin-4 (Cec4), which we successfully identified previously. Our results demonstrate that Cec4 not only exhibits rapid antibacterial activity but also effectively inhibits and eradicates bacterial biofilm at a low concentration of 8 µg/mL. Additionally, when used in combination with traditional antibiotics, Cec4 enhances their antibacterial effect. Microscopy techniques, including transmission electron microscopy (TEM), confocal laser scanning microscopy, and scanning electron microscopy (SEM), found that Cec4 destroyed bacteria's cell membrane integrity and increased the membrane permeability (flow cytometry instrument technology further characterization of Cec4 against bacteria antibacterial effect). Furthermore, experiments demonstrated that Cec4 binds to bacterial DNA and RNA of CRKP. Moreover, studies using a mouse skin wound model confirmed the efficacy of Cec4, and transcriptomic analysis shed light on the molecular mechanisms underlying its antibacterial activity. Based on our findings, Cec4 appears to be a promising candidate for combating CRKP infections.IMPORTANCEThe rapid increase in carbapenem-resistant (CRKP) infections and the serious cross-resistance to multiple antibiotics make the development of new therapeutic drugs urgent. Antimicrobial peptides (AMPs) have attracted much attention as a potential option for the next generation of antibiotics. Previous studies have identified the antimicrobial peptide Cecropin-4 (Cec4), and this study further explored its antimicrobial mechanism against CRKP. Studies have found that Cec4 shows high antibacterial activity at low concentrations, can inhibit and eradicate bacterial biofilms, and can also enhance the efficacy of traditional antibiotics. Its mechanism of action, such as destroying cell membranes and binding nucleic acid, has been revealed by various techniques, and its effectiveness has been confirmed , providing a promising candidate drug for combating CRKP infection.