Two isoforms of piscidin from Malabar grouper (Epinephelus malabaricus), EmPis-1 and EmPis-2, were cloned and studied. EmPis-1 and EmPis-2 showed the different in the 3′UTR features of mRNA and gene expression patterns. AUUUA-motif-containing ARE was found in mRNA of EmPis-1, but not in that of EmPis-2. EmPis-1 and EmPis-2 expressed not only in the potential sites of pathogen entry, but also in grouper's immune-related tissues such as head kidney (HD), peripheral blood leukocytes (PBL) and spleen. The expression level of EmPis-1 was higher than that of EmPis-2 in most fish tissues. Expression of both EmPis-1 and EmPis-2 were upregulated by V. parahaemolyticus significantly in the PBL, HD and spleen. Besides, expression of EmPis-1 was upregulated in gills. The putative mature peptides of EmPis-1 and EmPis-2, which were predicted to adopt an amphipathic α-helical conformation, posessed excellent microbicidal activities against both gram-negative and -positive bacteria. The hemolytic activity of the putative mature peptides of EmPis-1 and EmPis-2 increased in a dose-dependent manner to both grouper erythrocytes and rabbit erythrocytes. Interestingly, grouper erythrocytes were less vulnerable than rabbit erythrocytes to the peptides. Grouper piscidins excluded the signal peptide were not the inactive precursors but possessed high microbicidal activity evidenced by minimum bactericidal concentration (MBC) assay and by the scanning electron microscope (SEM) observation. The present phylogenetic analysis did not support the suggestion that piscidins are ancient AMPs widespread across invertebrate and vertebrate taxa, and that piscidins are included in the cecropin superfamily. Collectively, the present data improve our understanding of the piscidin family, and give greater insights into EmPis-1 and EmPis-2 of the grouper immune system.