The telomerase-specific replication-competent oncolytic adenovirus, Telomelysin, was developed for virus-mediated preferential lysis of tumor cells. Its selectivity is derived from a human telomerase reverse transcriptase (hTERT) promoter-driven active viral replication, which occurs in cancer cells with high telomerase activity but not in normal cells lacking such activity. Because the TERT activity is elevated in most cases of hepatocellular carcinoma (HCC), the current study aims to investigate whether Telomelysin can be used for treatment of HCC. The oncolytic effect of Telomelysin has been investigated both in vitro using cell culture and in vivo using an immunocompetent in situ orthotopic HCC model. In this model, HCC developed spontaneously in the liver of HBx transgenic mice, which is pathologically and genetically similar to human HCC. In cell culture assay, Telomelysin lyses HCC cell lines at a low multiplicity of infection (MOI), ranging 0.77-6.35 (MOI [PFU/cell]). In the orthotopic HCC model, Telomelysin showed a potent oncolytic effect on HCC but spared normal liver tissue. Dose escalation analysis identified a safety dose of 1.25 X 108 PFU for this model. The effect of multiple injections of Telomelysin was also evaluated in this immunocompetent HCC model. We found that the virus replicates in HCC after a second intratumoral injection despite an immune response induced by the previous injection. This preclinical study shows that Telomelysin can be used for treatment of human HCC at an appropriate dosage and that its tumor-killing activity persists after multiple injections.