The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.