Natural killer (NK) cells are considered a promising strategy for cancer treatment. Various methods for large-scale NK cell expansion have been developed, but they should guarantee that no viable cells are mixed with the expanded NK cells because most methods involve cancer cells or genetically modified cells as feeder cells. We used an anti-CD16 monoclonal antibody (mAb) and irradiated autologous peripheral blood mononuclear cells (PBMCs) (IrAPs) to provide a suitable environment (activating receptor-ligand interactions) for the NK cell expansion. This method more potently expanded NK cells, and the final product was composed of highly purified NK cells with lesser T-cell contamination. The expanded NK cells showed greater upregulation of various activation receptors, CD107a, and secreted larger amounts of interferon gamma. IrAPs expressed NKG2D ligands and CD48, and coengagement of CD16 with NKG2D and 2B4 caused potent NK cell activation and proliferation. The expanded NK cells were cytotoxic toward various cancer cells in vitro and in vivo. Moreover, irradiation or a chemotherapeutic drug further enhanced this antitumor effect. Therefore, we developed an effective in vitro culture method for large-scale expansion of highly purified cytotoxic NK cells with potent antitumor activity using IrAPs instead of cancer cell-based feeder cells.