The presence of regulatory T cells in patients who received therapeutic cytokine-induced killer (CIK) cells may inhibit host immunity, leading to failed immunotherapy. In this study, we investigated the impact of using interleukin-6 (IL-6) on the phenotype alteration, proliferation, and cytotoxic activity of CIK cells generated from the peripheral blood mononuclear cells of patients with hepatocellular carcinoma. We found that addition of IL-6 to CIK-cell culture medium decreased the percentage of Treg/CD4(+), Treg/CD3(+) T cells in the resultant CIK cells and simultaneously increased the proliferation ability, the expression of CD45RO(+)CD62L(low)CCR7(low) effector memory phenotype, and cytotoxicity of the CIK cells against hepatocellular carcinoma in vitro. Our results also showed that the percentage of Th17/CD4(+) cells was increased in CIK cells, but the proportion of Th17/CD4(+) cells was not affected by the addition of IL-6 to CIK-cell culture medium. Collectively, these data suggest that IL-6 may have the potential to improve antitumor activity of CIK cells in cancer immunotherapy.