CD137 (4-1BB) is a TNFR superfamily member that mediates the costimulatory signal resulting in T cells and NK cells proliferation and cytokines production, but the effects of CD137 signaling on CD3(+)CD56(+) cell subpopulation have not been well-documented. The aim of this study was to investigate the effects of CD137 signaling on regulation of CD3(+)CD56(+) cell function. Anti-CD137 mAb or mouse IgG1 isotype control was added to CIK cell culture to determine the effects of proliferation and anti-tumor effects on CD3(+)CD56(+) cells. We observed that anti-CD137 mAb could dramatically promote proliferation of CIK cells. And CD137-CIK cells and CD3(+)CD56(+) cell subpopulation within them possessed higher ability to kill tumor cell line A549. The SCID mice engrafted with A549 cells and treated with CD137-CIK cells have prolonged survival. Further studies revealed that the percentages of CD3(+)CD56(+) cells were elevated significantly in CD137-CIK cells. The expression of NKG2D was up-regulated on CD3(+)CD56(+) cells from CD137-CIK cells. The expression of IFN-gamma, IL-2 and TNF-alpha increased significantly whereas the production of TGF-beta(1), IL-4 and IL-10 decreased in CD3(+)CD56(+) cells from CD137-CIK cells. In addition, anti-CD137 mAb can elevate the capacity of CD3(+)CD56(+) cells to induce CD4(+) Th1 responses. We further showed that the anti-CD137 mAb also had the same effects on CD3(+)CD56(+) cells expanded from the PBMCs of patients with NSCLC. We concluded that CD137 signaling could enhance the abilities of CIK cells to kill tumor cells in vitro and in vivo via increasing the proportion of CD3(+)CD56(+) cells and their cytotoxicity. Furthermore, CD137 signaling can elevate the capacity of CD3(+)CD56(+) cells to induce CD4(+) Th1 responses which may enhance their anti-tumor activity indirectly. Taken together, our studies could be considered as valuable in CIK cells-based cancer immunotherapy.