HBx is an oncogenic tumor-associated antigen and is dominantly expressed in hepatitis and hepatoma tissues, the induction of active cellular responses against HBx should be a promising approach for the treatment of hepatitis B virus-related hepatocellular carcinoma. The present study was designed to test whether a replication-defective adenovirus vaccine expressing HBx antigen could be effectively used in the immunotherapy of hepatocellular carcinoma. To validate the possibility, we developed a novel HBx-positive hepatocellular carcinoma in mice by inoculated the pcDNA-HBx transfected Hepa1-6 cells subcutaneously into the right flank of mice. We found that immunotherapy with Ad-HBx was effective at both protective and therapeutic antitumor immunity in the hepatoma models in immune-competent mice. Histological examination revealed that Ad-HBx treatment led to significantly increased induction of apoptosis, tumor necrosis, and elevated CD8+ lymphocyte infiltration. In addition, the induction efficacy of the CTL response is dramatically enhanced by immunotherapy. Cytokine analysis confirmed that the antitumor efficacy of Ad-HBx may mostly result from cellular immunity. Our findings may prove useful in development of adenovirus vaccine based on HBx antigen to the treatment of HBV-associated hepatocellular carcinoma.