Natural killer (NK) cell adoptive transfer is a promising approach for cancer immunotherapy; however, its development has been hindered by the lack of efficient methods to produce large numbers of functional NK cells. In this study, we engineered the leukaemia cell line K562 to express CD137 ligand (CD137L) and membrane-bound interleukin (mbIL)-21 on the cell surface, and used these cells to expand NK cells from the peripheral blood mononuclear cells. We found that purity of the NK cells (CD3−CD56+/CD16+) increased from less than 30% to above 95% after a 3-week expansion and proliferation of the cells was sustained for more than 8 weeks. The surface expression of NK cell activating and inhibitory receptors, except for NKp80, was clearly increased with the expansion, and NK cell-mediated killing activity was also enhanced significantly. However, these changes in both phenotype and function were clearly reversed by JSI-124, a specific signal transducer and activator of transcription-3 (STAT-3) inhibitor. Taken together, data showed that the combination of mbIL-21 and CD137L could efficiently induce the formation of functional human NK cells from peripheral blood mononuclear cells, and STAT-3 inhibition could impair this induction. Therefore, STAT-3 activation may benefit human NK cell proliferation and cytotoxicity, and provide valuable clinical applications in NK cell immunotherapy against viral infectious diseases and cancers.