Natural killer cells are lymphocytes of the innate immune system that play a key role in the direct elimination of transformed or virus-infected cells. Recently, it has been reported that NK cells can attack cancer cells with stem cell-like properties. In this study, we isolated ovarian cancer cell lines CAOV3 and TOV21G with and without CD24, which has been reported as an ovarian cancer stem cell marker, and compared their drug resistance and susceptibility to NK cell lysis. The isolated CD24(+) CAOV3 and TOV21G cells were more resistant to cisplatin and doxorubicin anti-cancer drugs. Also, CD24(+) CAOV3 and TOV21G cells were more susceptible to NK cell lysis compared with CD24(-) cells. In order to identify reasons for the differing NK cell susceptibility, we examined NK cell-killing mechanisms against CD24(+) cancer cell lines by analyzing NKG2D ligands, MHC class I molecules, and natural cytotoxic receptor ligands expression on target cells. Consistently, CD24(+) CAOV3 and TOV21G cells showed up-regulated NKG2D ligands and down-regulated MHC class I molecule expression. These findings show that CD24(+) ovarian cancer cell lines are more resistant to antitumor drugs but are more susceptible to NK cell lysis; thus, NK cell immunotherapy might be useful in eliminating ovarian cancer stem cells and preventing tumor recurrence and metastasis.