Dendritic cell (DC) vaccine is a potent immunotherapeutic approach for cancer treatment, but the clinical efficacy needs to be improved. In this study, we evaluated the combinational effect of Toll-like receptor 7 (TLR7) agonist Imiquimod and BM-DC vaccine against mouse melanoma and explored the potential mechanisms. We found that topical application of Imiquimod cream caused skin inflammation and enhanced exogenous BM-DC homing to draining lymph nodes. Imiquimod treatment enhanced DC vaccine efficacy against B16-OVA melanoma. The combinational modality enhanced cytotoxicity of splenic lymphocyte to tumor cells and inhibited CD4(+)FOXP3(+)Treg cell production. TLR7 mRNA expression was confirmed in both MC/9 mast cells and DCs. MC/9 cells treated by R837 (soluble form of Imiquimod) enhanced CD80, CD86, MHC-II and CCR7 expression on DCs. R837 inhibited B16-OVA cell growth in vitro. Our findings suggest that Imiquimod can be used as a potent adjuvant in the formulation of a DC-based tumor fighting vaccine. The mechanisms underlying these effects of Imiquimod are related with enhanced DC homing to DLNs, inhibition of Treg's production, direct tumor cell toxicity and synergistic function with mast cell in enhancing DC activation.