Natural killer (NK) cells are promising antitumor effector cells, but the generation of sufficient NK cell numbers for adoptive immunotherapy remains challenging. Therefore, we developed a method for highly efficient expansion of human NK cells. expansion of NK cells in medium containing IL-2 and irradiated clinical-grade feeder cells (EBV-LCL) induced a 22-fold NK cell expansion after one week that was significantly increased to 53-fold by IL-21. Repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 10-fold NK cell expansion after 6 weeks. Compared to naive NK cells, expanded NK cells upregulated TRAIL, NKG2D, and DNAM-1, had superior cytotoxicity against tumor cell lines and produced more IFNγ and TNF-α upon PMA/Iono stimulation. Most importantly, adoptive transfer of NK cells expanded using feeder cells, IL-2 and IL-21 led to significant inhibition of tumor growth in a melanoma xenograft mouse model, which was greater than with NK cells activated with IL-2 alone. Intriguingly, adoptively transferred NK cells maintained their enhanced production of IFNγ and TNF-α upon restimulation, although they rapidly lost their capacity to degranulate and mediate tumor cytotoxicity after the transfer. In conclusion, we developed a protocol for NK cell expansion that results in outstanding cell yields. The expanded NK cells possess potent antitumor activity and and could be utilized at high numbers for adoptive immunotherapy in the clinic.