One of the major limitations for cancer immunotherapy is related to the frequent existence of an intra-tumoral immunosuppressive environment, to which STAT3 (Signal transducer and activator of transcription-3) activation in tumor and dendritic cells (DCs) are believed to contribute. In this study, we tested the hypothesis that the combination of CpG (a DC activator) and JSI-124 (a STAT3 inhibitor) may generate synergistic antitumor effects compared to CpG or JSI-124 alone. B16-F10, a mouse melanoma cell line that has constitutively active STAT3, was grafted in C57BL/6 mice and then tumor-bearing mice treated intra-tumorally with (a) phosphate buffered saline, (b) 10 microg CpG, (c) 1 mg kg(-1) JSI-124 or (d) 10 microg CpG+1 mg kg(-1) JSI-124. The effects of treatments on tumor growth, survival and antitumor immune responses were evaluated. Although significant antitumor effects were detected with the single-agent treatments, the CpG+JSI-124 treatment resulted in synergistic antitumor effects compared to CpG or JSI-124 alone. Correlating with these findings, the combination therapy resulted in significantly higher intra-tumoral levels of several proinflammatory, TH1-related cytokines (including IL-12, IFN-gamma, TNF-alpha and IL-2), increases in intra-tumoral CD8+ and CD4+ T cells expressing activation/memory markers and NK cells and increases in activated DCs in the tumors and regional lymph nodes (LNs). Concomitantly, the combination therapy led to a significantly decreased level of immunosuppression, as evidenced by lower intra-tumoral level of VEGF and TGF-beta, and decreased number of CD4+CD25+Foxp3+ regulatory T cells in the regional LNs. This study has provided the proof-of-principle for combining CpG and JSI-124 to enhance antitumor immune responses.