The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. For some patients for whom surgical treatments are not appropriate, one can only rely on chemotherapy. In the conventional chemotherapy, side effects usually occurred in most cases due to high toxicity levels. Moreover, the development of drug resistance toward chemotherapeutic agents often prevents the successful long-term use of chemotherapy for HCC. Gene therapy represents the exciting biotechnological advance that may revolutionize the conventional fashion of cancer treatment. Overexpression of phosphatase and tensin homologue (PTEN) in cancer cells carrying deletion/mutant type of it can induce the apoptosis of cancer cells and inhibit cell proliferation. In this work, in order to make full use of the high transfectivity of adenovirus, we managed to conjugate the polysaccharide mannan (polymannose) to the surface of the adenovirus chemically under appropriate oxidizing conditions to prepare the mannan-modified adenovirus (Man-Ad5-PTEN). The cytotoxicity and anticancer activity of Man-Ad5-PTEN were assessed in vitro. Reporter gene expression of LacZ transferred by Man-Ad5-LacZ was verified on mannose receptor-deficient NIH/3T3 cells versus mannose receptor-efficient macrophages. Hepatocellular carcinoma cell lines transduced by mannan-modified adenovirus were assayed for cell cycle, apoptosis, invasion, and migration. Further, we detected the antitumor effect on intraperitoneal H22 tumor-bearing mice treated by Man-Ad5-PTEN alone or combined with chemotherapeutic agent of doxorubicin. The results demonstrated that cell growth suppression was not observed in Chang normal hepatocyte cells and the cell killing by Man-Ad5-PTEN is tumor selective. Further, the results showed that the strategy of mannan conjugation could enhance adenovirus-mediated PTEN gene therapy effects on murine hepatocellular carcinoma cells in vitro and in vivo.