Regulatory T cells (Tregs) are potent immunosuppressive cells that promote tumor growth and invasion by inducing immune escape and suppressing the antitumor immune response. Cytokine-induced killer (CIK) cells are considered to be the primary candidate for adoptive immunotherapy due to their strong antitumor activity. It was recently reported that the concomitant presence of Tregs may decrease the cytotoxicity of CIK cells. Therefore, depletion or downregulation of Tregs in CIK cell cultures by optimizing the culture program may enhance CIK cell cytotoxicity in vitro and in vivo. The aim of the present review was to summarize the currently available studies on the optimal culture strategy for improving the antitumor activity of CIK cells through targeting Tregs.