A large number of natural killer (NK) cells with high function are expected to generate especially in tumor adoptive immunotherapy. Here K562 cells were genetically modified to co-express major histocompatibility complex class I chain-related protein A (MICA), 4-1BB ligand, and IL-15, called K562-MICA-4-1BBL-IL-15. The modified K562 cells not only promoted activation, proliferation, and survival of NK cells, but also enhanced NK cell cytotoxicity. In long-term culture tests, K562-MICA-4-1BBL-IL-15 cells stimulated NK cell to expand mean 550 folds in 24-day culture and to cover from 14.8% of total peripheral blood monoclonal lymphocytes on day 1 to 86.7% on day 24. Prevalent NK cells after expansion enhanced the ability of killing targets and producing interferon gamma (IFN-γ), and kept high expression of activating receptors. The results indicated that K562-MICA-4-1BBL-IL-15 cells would be developed for expansion of NK cells ex vivo and may have important implications for clinical immunotherapy.