The effect of IFN-α on the immunosuppressive tumor microenvironment is not fully understood. We previously reported that intratumoral IFN-α gene transduction decreased the frequency of regulatory T cells (Tregs) in the tumor by inducing the secretion of IL-6 from dendritic cells. In this study, we examined whether IFN-α affects the trafficking of Tregs to the tumor. Since CT26 cells expressed CCL17 among Treg-attracting chemokines, we focused on its role in IFN-α-mediated Treg suppression. IFN-α directly suppressed CCL17 production from CT26 cells in vitro, and IFN-α transduction reduced CCL17 expression in tumors in vivo. Next, to investigate whether CCL17 downregulation is related to the suppression of Treg trafficking, CCL17-downregulated CT26 cells produced using short hairpin RNA (CT26-shCCL17) were inoculated into mice. The frequency of Tregs in CT26-shCCL17 tumors was reduced and tumor growth was suppressed. Finally, to examine the combinatorial effect of IFN-α expression with CCL17 downregulation, IFN-α was transduced into CT26-shCCL17 tumors. This resulted in an elevation of CT26-specific CD8+ T cells and the complete eradication of tumors. This study shows a novel mechanism of IFN-α-mediated Treg suppression, and combining IFN-α gene therapy with strong CCL17 downregulation could offer a promising strategy for the treatment of cancer.