BACKGROUND: Natural killer (NK) cells are recognized for their ability to kill tumor cells for tumor control, but tumor cells often develop resistance to evade NK cell-mediated cytotoxicity. Identification of molecular mechanisms by which tumor cells evade from NK cell-mediated killing may offer novel therapeutic strategies for potentiating NK-based cancer immunotherapy. METHODS: An in vitro tumor-NK cell co-culture system was employed to identify the most significantly altered genes in tumor cells following NK cell interaction. The cell death rate of tumor cells by NK cell exposure was quantified using flow cytometry. EL4 and HCT116 tumor models in C57BL/6, BALB/c-nu, and NOD/SCID mice were used for evaluating tumor growth differences induced by knockdown or knockout. The cellular and molecular impact of knockdown or knockout on the sensitivity of tumor cells to NK cell-mediated cytotoxicity was assessed using quantitative PCR, immunofluorescence, and mutation analysis. RESULTS: By screening expression levels of the Ras homology (Rho) GTPase family genes in tumor cells after co-culture with NK cells, we identified RAC2 as a key regulator of tumor cell resistance to NK cell-mediated cytotoxicity among the Rho GTPase family members. Furthermore, knockout of in human colorectal cancer cells leads to increased tumor susceptibility to NK cell-mediated cytotoxicity in a xenograft tumor model. Mechanistically, the absence of RAC2 enhances tumor cell sensitivity to NK cell-mediated killing by facilitating cell-cell contact. CONCLUSIONS: These findings indicate that the inhibition of RAC2 in tumor cells substantially enhances their susceptibility to NK cell-mediated cytotoxicity, thereby providing a potential therapeutic target for optimizing NK cell therapy.