NK-lysin, an effector of cytotoxic T cells and natural killer cells, is a potent antimicrobial peptide widely distributed in mammals. Homologues of NK-lysin have been discovered in several teleost species, but only several of their natural functions was recorded so far. Here we identified an NK-lysin from Nile tilapia (Oreochromis niloticus), On-NKL, and analysed its expression model and biological effects on pathogen infection. The open reading frame of On-NKL sequence spans 432 bp, codes for 143 amino acids and shares 27%–62% overall sequence identities with NK-lysin of other species. The deduced mature peptide of On-NKL possesses a saposin B domain and six well-conserved cysteine residues that essential for antimicrobial activity by forming three intrachain disulphide bonds. The results of qRT-PCR showed that On-NKL expression was observed in multiple tissues and head kidney leucocytes and nonspecific cytotoxic cells (NCCs) and is most abundant in gills. After bacterial challenge, On-NKL expression significantly varied in different tissues and NCCs. Following bacterial infection, On-NKL-overexpressing fish featured significantly lower pathogen loads in tissues than control fish. On-NKL-overexpressing fish also exhibited 33.3% relative percent survival compare with control groups. Findings suggested that On-NKL could be the potential effector of NCCs and act as immune-related gene that enhances antimicrobial defence.