The generation of genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Cytokine-induced killer cells (CIKs) have been described as highly efficient cytotoxic effector cells capable of recognizing and lysing tumor cell targets in a non-major histocompatibility complex restricted fashion. In the present study, we evaluated the effects of inducible costimulator (ICOS) on the cytotoxicity of CIK cells against gallbladder cancer. We first prepared CIK-ICOS cells by the transfection of ICOS genes into induced CIK cells, whereas untransfected or enhanced green fluorescent protein (EGFP)-transfected CIK cells were treated as controls. We found that CIK-ICOS cells displayed better proliferation and lower apoptosis than the other two CIK control cells after culture. The interferon-gamma level in the culture supernatant of CIK-ICOS cells was also significantly elevated, compared to CIK or CIK-EGFP cells. The cytotoxic effect of CIK-ICOS cells against gallbladder cancer cells was dramatically enhanced at the E:T ratio of 20:1, compared to that of CIK or CIK-EGFP cells. When injected into gallbladder tumor-bearing SCID mice, CIK-ICOS cells significantly slowed down the growth rate of xenografts. CIK-ICOS-treated mice exhibited the least volume variation of the xenografts and more severe necrosis, compared to saline, CIK, or CIK-EGFP cell-treated mice, accompanied by a better in situ survival around xenografts than the other two control cells. Taken together, our results demonstrated that ICOS could enhance the cytotoxic activity of CIK cells, in part, by augmenting cytokine secretion and prolonging cell survival both in vitro and in vivo. This might be considered as the potential immunomodulator for clinical therapy. Copyright © Informa Healthcare USA, Inc.