Accumulating evidence has indicated that preconditioning chemotherapy could eliminate the suppressive factors in antitumor immune response, thereby leading to the full release of the efficacy of the subsequent immunotherapy. In this study, a single subtoxic dose (5 mg/kg, intraperitoneally) of cisplatin was chosen as the preconditioning chemotherapy in combination with cytokine-induced killer (CIK) cells (4×10(6), intravenously) to treat the murine B16 melanoma xenografts. It was found that cisplatin pretreatment could enhance the antitumor activity of CIK cells. To explore the potential mechanisms underlying the efficacy-enhancing effect of cisplatin, the in vivo trafficking and distribution of the infused CIK cells were traced. It was found that cisplatin could augment the homing ability of CIK cells into the tumor, tumor-draining lymph nodes (TDLNs), and spleen tissues. The endogenous effector cells, CD3(+) T lymphocytes also had an increased accumulation in the tumor and TDLNs after cisplatin precondition. Moreover, cisplatin could also modulate the percentages of myeloid cells, thus encouraging immune responses by increasing the percentages of dendritic cells and relieving the immunosuppression by preferentially eliminating the myeloid-derived suppressor cells. In conclusion, our findings suggested that cisplatin preconditioning chemotherapy could enhance the antitumor activity of CIK cells in a murine melanoma model, and this efficacy-enhancing effect was attributed to the augmented homing ability of exogenous and endogenous effector cells and the modulation of the myeloid cells.