Antimicrobial peptides (AMPs) play important roles in innate immunity. One such AMP, epinecidin-1, exhibits antibacterial effects in zebrafish. In the current study, we aimed to identify the antimicrobial-associated proteins affected by epinecidin-1 treatment, and to unravel the underlying antimicrobial molecular mechanisms of epinecidin-1. We analyzed proteome changes in epinecidin-1-treated zebrafish using two-dimensional electrophoresis (2DE) coupled to mass spectrometry. Several differentially expressed proteins were identified, some of which were validated by real-time quantitative RT-PCR. The differentially expressed proteins were mapped onto Ingenuity Pathway Analysis canonical pathways, to construct a possible protein–protein interacting network regulated by epinecidin-1; this network suggested a potential role of epinecindin-1 in cytoskeletal assembly and organization. Our findings imply that epinecidin-1 may stabilize the cytoskeleton network in host cells, thereby promoting resistance to bacterial infection.