Natural killer (NK) cells play an important role in anti-tumor immunity. Interleukin (IL)-18 is animmunoregulatory cytokine that induces potent NK cell-dependent anti-tumor responses when administratedwith other cytokines. In this study, we explored the effects of combining IL-18 and IL-2 on NK cytotoxicity aswell as expression levels of the NK cell receptor NKG2D in vitro. Freshly isolated PBMCs were incubated for48 h with IL-18 and IL-2, then CD107a expression on CD3-CD56+ NK cells was determined by three-colour flowcytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human coloncarcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on NK cells.The combined use of IL-18 and IL-2 significantly increased CD107a expression on NK cells compared with usingIL-18 or IL-2 alone, suggesting that the combination of these two cytokines exerted synergistic enhancement ofNK cytotoxicity. IL-18 also enhanced NKG2D expression on NK cells when administered with IL-2. In addition,blockade of NKG2D signaling with NKG2D-blocking antibody attenuated the up-regulatory effect of combiningIL-18 and IL-2 on NK cytolysis. Our data revealed that IL-18 synergized with IL-2 to dramatically enhance thecytolytic activity of human NK cells in a NKG2D-dependent manner. The results appear encouraging for theuse of combined IL-18 and IL-2 in tumor immunotherapy.