Natural killer (NK) cell-based immunotherapy is promising, as NK cells are in the first line of defense against cancer and capital of lysing tumor cells without pre-stimulation. However, NK cells from multiple myeloma (MM) patients are always deficient in numbers and the expression of certain activating receptors, disabling them in cytotoxicity against the cancer. Therefore, effective strategies to expand NK cells and increase NK cell-mediated cytotoxicity against MM are significant. Here, NK cells were efficiently expanded from peripheral blood mononuclear cells (PBMCs) of newly diagnosed MM patients after co-culture with irradiated K562 cells transfected with 41BBL and membrane-bound interleukin (IL)-15 (K562-mb15-41BBL) in the presence of 200 IU/ml human IL-2. The ex vivo-expanded NK cells were demonstrated to vigorously kill both MM cells and autologous primary MM cells without significant lysis of patient normal cells. Further exploration revealed a significant increase in cell surface expression of most activating receptors of NK cells and indicated that expanded NK (exp-NK) cell killing of MM cells was mediated by perforin/granzyme. NK cells are capital of lysing human leukocyte antigen (HLA) I-deficient tumor cells and carfizomib, a selective proteasome inhibitor approved for the treatment of relapsed/refractory MM patient, down-regulates the expression of HLA class I, thus enhancing NK cell-mediated lysis in MM. Here, we found for the first time that carfizomib dramatically augmented ex vivo exp-NK cell cytotoxicity against patient autologous MM cells, suggesting the use of exp-NK alone or in combination with the drug to treat MM patient.