Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy. However, clinical trials have indicated that immunosuppressive microenvironments induced by tumors profoundly suppress antitumor immunity and inhibit vaccine efficacy, resulting in insufficient reduction of tumor burdens. To overcome these obstacles and enhance the efficiency of DC vaccination, we generated interleukin (IL)-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-coexpressing oncolytic adenovirus (Ad-ΔB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-ΔB7/IL12/GMCSF prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CC-chemokine ligand 21 (CCL21(+)) lymphatics in tumors treated with Ad-ΔB7/IL12/GMCSF. Moreover, the proportion of CD4(+)CD25(+) T-cells and vascular endothelial growth factor (VEGF) expression was decreased in mice treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-ΔB7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efficacy on large tumors. Taken together, oncolytic adenovirus coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination.