Hepatocellular carcinoma (HCC) is one of the cancers with the highest morbidity and mortality in the world. However, clinical progress in the treatment of HCC has not shown a satisfactory therapeutic effect. Here, we have developed a novel strategy to treat HCC with an adenovirus (Ad)-based vaccine, which contains a specific antigen glypican-3 (GPC3) and an immunostimulatory cytokine IL-12. In the subcutaneous tumor model, Ad-IL-12/GPC3 vaccine was injected into muscles three times to evaluate its therapeutic effect. Compared with the control immunization group, the Ad-IL-12/GPC3 immunization group showed a significant tumor growth inhibition effect, which was confirmed by the reduced tumor volume and the increased tumor inhibition. Ad-IL-12/GPC3 co-immunization promoted the induction and maturation of CD11c or CD8CD11c DCs and increased the number of tumor-infiltrating CD8 T cells. Furthermore, in the Ad-IL-12/GPC3 group, the proliferation of CD8 T cells, the induction of multifunctional CD8 T cells, and CTL activity were significantly increased. Interestingly, the deletion of CD8 T cells abolished tumor growth inhibition by Ad-IL-12/GPC3 treatment, suggesting that CD8 T cell immune responses were required to eliminate the tumor. Likewise, Ad-IL-12/GPC3 vaccine also effectively inhibited lung tumor growth or metastasis by enhancing CD8 DCs-mediated multifunctional CD8 T cell immune responses in the lung metastasis model. Therefore, these results indicate that IL-12 combined with Ad-GPC3 vaccine co-immunization might provide a promising therapeutic strategy for HCC patients.