Tregs are potent immunosuppressive cells and essential for inducing immune tolerance.Recent studies have reported that Tregs and Tregs related cytokines can inhibit the antitumor activity of CIK cells, but DC-CIK cells can be used for induction of a specific immune response by blocking of Tregs and TGF-β,IL-10.As a novel identified cytokine, IL-35 is specially produced by Tregs and plays an essential role in immune regulation.However, it remains unknown whether IL-35 roles in tumor immunotherapy mediated by CIK and DC-CIK cells.In this study, we cultured CIK and DC-CIK cells from the same healthy adult samples, and investigated their phenotype, proliferation, cytotoxic activity against leukemia cell lines K562 and NB4 by FCM and CCK-8, measured IL-35, TGF-β and IL-10 protein by ELISA, detected Foxp3, IL-35 and IL-35 receptor mRNA by Real-time PCR, respectively.We found Tregs and IL-35 concomitantly expanded by a time-dependent way during the generation of CIK cells, but DC significantly down-regulated the expression of them and simultaneously up-regulated the proliferation ability as well as cytotoxic activity of CIK cells against leukemia cell lines.Therefore, our data suggested that DC decreased concomitant expanded Tregs and Tregs related IL-35 in CIK cells and might contribute to improve their cytotoxicity against leukemia cells in vitro.