Vibrio parahaemolyticus is one of the most common pathogenic bacteria that pose a threat to human health. The purpose of this study was to investigate antibacterial mechanisms of ε-poly-lysine (ε-PL) against V. parahaemolyticus using a lable free-based proteomic analysis. The differentially expressed proteins (DEPs) were subjected to bioinformatics analysis. The results indicated that a total of 196 DEPs, including 118 up-regulated and 78 down-regulated, were identified in the ε-PL-treated cells compared with control group. Upon Go functional enrichment, 13, 9, and 8 specific Go terms in biological processes, molecular functions and cellular components were identified, respectively. KEGG pathways analysis indicated that the DEPs were mainly involved in bacterial chemotaxis, RNA transport and two-component system, which were significantly enriched (P < 0.05). In PPI analysis, Che R and Che V, both involved in bacterial chemotaxis and RNA transport pathways, are closely related to other DEPs. Therefore, the down-regulation of Che R and Che V in ε-PL-treated cells resulted in the reduction or even loss of bacterial adaptability, and they were the critical action sites of ε-PL to inactivate V. parahaemolyticus.