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Antimicrobial peptides (AMPs), most of which are small proteins, are necessary for innate immunity against pathogens. Anti-lipopolysaccharide factor (ALF) with a conserved lipopolysaccharide binding domain (LBD) can bind to lipopolysaccharide (LPS) and neutralize LPS activity. The antibacterial mechanism of ALF, especially its role in bacteria, needs to be further investigated. In this study, the antibacterial role of an anti-lipopolysaccharide factor (PcALF5) derived from Procambarus clarkii was analyzed. PcALF5 could inhibit the replication of the microbiota in vitro and enhance the bacterial clearance ability in crayfish in vivo. Far-western blot assay results indicated that PcALF5 bound to two proteins of E. coli (approximately 25 kDa and 15 kDa). Mass spectrometry (MS), far-western blot assay, and pull-down results showed that 30S ribosomal protein S4 (RPS4, 25 kD) interacted with PcALF5. Further studies revealed that another E. coli protein binding to PcALF5 could be the large mechanosensitive channel (MscL), which is reported to participate in the transport of peptides and antibiotics. Additional assays showed that PcALF5 inhibited protein synthesis and promoted the transcription of ribosomal component genes in E. coli. Overall, these results indicate that PcALF5 could transfer into E. coli by binding to MscL and inhibit protein synthesis by interacting with RPS4. This study reveals the mechanism underlying ALF involvement in the antibacterial immune response and provides a new reference for the research on antibacterial drugs.