NK-lysin is a cationic antimicrobial peptide mainly produced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and stored in cytolytic granules together with perforin and granzymes. NK-lysin is a member of saposin-like protein family and is orthologous with human granulysin. Mammalian NK-lysin is known to possess antibacterial and antitumor property. Homologues of NK-lysin have been identified in diverse organisms including human, pig, cattle, chicken, horse, water buffalo and teleost species. In fish, NK-lysin genes have been reported in five species, i.e. Japanese flounder (Paralichthys olivaceus), channel catfish (Ictalurus punctatus), zebrafish (Danio rerio), half-smooth tongue sole (Cynoglossus semilaevis) and large yellow croaker (Larimichthys crocea). In this study, we aimed to explore the structure and expression difference in tissues, and the NK-lysin (Cinkl) was identified from grass carp Ctenopharyngodon idella using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE), and primers were designed according to the conserved sequence of known fish NK-lysin genes. The expression of the Cinkl gene in different tissues was analyzed by real-time quantitative PCR. In order to explore the antibacterial activity of CiNkl, the recombinant expression vector was constructed by cloning its mature peptide into the prokaryotic expression plasmid pET-28b-MBP. The antibacterial analysis of recombinant protein was evaluated using agar disc diffusion. The full-length cDNA sequence of Cinkl was 768 bp, encoding 121 amino acids. Genomic DNA was 3 361 bp including 4 exons and 3 introns that was similar to zebrafish nklc and nkld genome. The multiple sequence alignment analysis showed that the CiNkl shared 57.98% and 63.03% identity with zebrafish Nklc and Nkld respectively. CiNkl possesses a Saposin B domain of members of saposin-like protein (SAPLIP) family and six conserved cysteins (Cys) residues that in mammals are known to form three intramolecular disulfide bonds essential to antimicrobial activity. Phylogenetic tree suggested that CiNkl is clustered closely with D. rerio Nklc and Nkld. Quantitative RT-PCR results showed that Cinkl mRNA was expressed in all tissues examined and highly in the spleen, with small amount in heart, gill, kidney, head kidney and weakly in skin, brain, liver and intestine. In addition, SDS-PAGE showed that the molecular mass of recombinant CiNkl expressed in host bacteria Rosetta (DE3) was approximately 57 ku. The recombinant protein purified by Ni-NTA His·Bind resin showed the antibacterial activity against Escherichia coli M15, Aeromonas hydrophila and Staphylococcus aureus. The results indicate that grass carp NK-lysin has potential immunomodulatory effects in fish innate immunity.