Background: Hepatocellular carcinoma (HCC) is one of the most difficult tumors to be treated. Overexpression of human telomerase reverse transcriptase (hTERT) and dysfunction of retinoblastoma (RB) have been implicated in HCC. Oncolytic viruses have been used as targeting therapy for different malignancies. In study, we have engineered oncolytic adenovirus to target both of TERT and RB pathways to achieve strict tumor specificity. Methods: A double-controlled recombinant adenovirus (AdCN103) was constructed by deletion of 24 bp in CR2 region of E1A and replacement of E1A promoter with the modified hTERT promoter. As control vectors, we generated single-controlled recombinant adenovirus with either 24 bp deleted E1A (AdCN101) or wild-type of E1A driven by hTERT promoter (AdCN102). Results: Our results showed that infection of HCC cell lines with AdCN101, AdCN102 or AdCN103 resulted in strong cytotoxicity. In contrast, there was a dramatic reduction of cytotoxicity in normal cells infected with AdCN102 and AdCN103. Production of viral particles in HCC cells infected with AdCN103 as measured by virus progeny assay was similar to cells infected with AdCN101 or AdCN102. A significant higher viral production was observed in 2 out of 4 normal cell line infected with AdCN102 than AdCN103. Furthermore, these vectors could repress the tumor growth and prolong survival in tumor-bearing animals with HCC. Conclusion: These dada indicated that oncolytic adenovirus to target both of TERT and RB pathways provided a safe and potent vector for the gene therapy of HCC. ©2007 Landes Bioscience.