The plasma cell malignancy, multiple myeloma (MM), has significantly improved by the application of new drugs and autologous hematopoietic stem cell transplantation. However, MM remains incurable. A number of studies have revealed an anti-MM effect of natural killer (NK) cells; however, their clinical efficacy is limited. Furthermore, glycogen synthase kinase (GSK)-3β inhibitors show an antitumor function. In this study, we aimed to evaluate the potential roles of a GSK-3β inhibitor (TWS119) in the regulation of NK cell cytotoxicity against MM. Our results showed that, in the presence of TWS119, the NK cell line, NK-92, and in vitro-expanded primary NK cells exhibited a significantly higher degranulation activity, expression of activating receptors, cellular cytotoxicity, and cytokine secretion when they were exposed to MM cells. Mechanistic studies indicated that TWS119 treatment markedly upregulated RAB27A expression, a key molecule for NK cell degranulation, and induced the colocalization of β-catenin with NF-κB in the nucleus of NK cells. More importantly, GSK-3β inhibition combined with the adoptive transfer of TWS119-treated NK-92 cells significantly reduced tumor volume and prolonged the survival time of myeloma-bearing mice. In summary, our novel findings suggest that targeting GSK-3β through the activation of β-catenin/NF-κB pathway may be an important approach to improve therapeutic efficacy of NK cell transfusion for MM.Keywords: GSK-3β; NF-κB; NK cells; immunotherapy; β-catenin.