Hedyotis diffusa is a well-known traditional Chinese herbal medicine. The polysaccharides extracted from H. diffusa (HDP) exhibit a range of pharmacological activities. Transfusion of cytokine-induced killer (CIK) cell is one type of adoptive cellular immunotherapy, which is becoming an important method of cancer immunotherapy. In this present study, we investigate the immunostimulatory effect of HDP on CIK cells. CIK cells were generated by culturing and stimulating peripheral blood monocytes of healthy volunteers. They were treated with HDP at three different concentrations (10, 50, and 100 μg/mL). The effect of HDP on CIK cell populations, intracellular cytokine production, and apoptosis was examined by flow cytometry. The antitumor effect of HDP on CIK cells was determined by cytotoxicity assay. Furthermore, the effect of HDP on the antitumor activity of CIK cells in a mouse model was investigated. HDP increased the percentage of CD3⁺CD56⁺ CIK cells but did not significantly change the percentage of CD4⁺, CD8⁺, or CD4⁺CD25⁺ CIK cells. The HDP-treated CIK cells showed a greater ability to kill tumor cells, as well as higher production of interferon-γ and tumor necrosis factor-α, compared with the no-HDP-treated CIK cells. The HDP-treated CIK cells also found a lower apoptosis level in vitro. Moreover, HDP combined with CIK cells had a stronger inhibitory effect on tumor growth in the mouse model compared with the CIK or HDP treatment alone. In conclusion, the results indicated that HDP enhanced the antitumor activity of CIK cells and could be used for cancer immunotherapy combined with CIK cell therapy.