NK-lysin is a component of natural killer cells and cytotoxic T lymphocytes, and it plays an important role in the innate immune response of aquatic animals against various pathogens. In this study, a 640 base pair full-length cDNA sequence of the NK-lysin gene from black sea bass (Centropristis striata) (CsNKl) was obtained and analyzed. Tissue distribution results showed that CsNKl was widely distributed in various tissues of C. striata, but a significantly high expression level was detected in the spleen. CsNKl also exhibited time-dependent expression in different tissues after challenge with Vibrio harveyi. Two antimicrobial peptides (CsNKl-26 and CsNKl-28) were predicted from CsNKl proteins, and they demonstrated bactericidal functions against a variety of bacteria. Scanning electron microscopy analysis revealed that CsNKl-26 and CsNKl-28 played their bactericidal roles using the classical alpha-helix antimicrobial peptide route. These results illustrate the immune functions of CsNKl against V. harveyi, and they can be applied to developing disease control methods for fish.