Description: Background: Oncolytic herpes simplex virus (HSV) can replicate in and kill cancer cells while sparing the adjacent normal tissue. Hepatocellular carcinoma (HCC) is amongst the most common and lethal cancers, especially in Third World countries. In this study, the cytotoxicity of a third-generation oncolytic HSV, G47?, was investigated in different human HCC cell lines and in an immortalized human hepatic cell line. Additionally, subcutaneous models of HCC were established to evaluate the in vivo anti-tumor efficacy of G47?. Methods: The HepG2, HepB, SMMC-7721, BEL-7404, and BEL-7405 human HCC cell lines and the HL-7702 human hepatic immortalized cell lines were infected with G47? at different multiplicities of infection (MOIs). The viability of infected cells was determined, and the G47? replication was identified by X-gal staining for LacZ expression. Two subcutaneous (s.c.) HCC tumor models of HCC were also established in Balb/c nude mice, which were intratumorally(i.t.) treated with either G47? or mock virus. Tumor volume and mouse survival times were documented. Results: More than 95% of the HepG2, Hep3B,and SMMC-7721 HCC cells were killed on by day 5 after infection with a MOI?s of 0.01. For the HL-7702 human hepatic immortalized cells, 100% of the cells were killed on by day 5 after infection with a MOI?s of 0.01. The BEL-7404 HCC cell line was less susceptible with about 70% cells were killed by day 5 after infection with a MOI?s of 0.01. Whereas the BEL-7405 HCC cells were the least susceptible, with only 30% of the cells were killed. Both the SMMC-7721 and BEL-7404 cells form aggressive sc tumor models. G47? replicates in the tumors, such that most of the tumors regressed after the G47?-treatment, and treated tumor-bearing mice survived much longer than the control animals. Conclusions: G47? effectively kills human HCC cells and an immortalized hepatic cell line at low MOI. Intra-tumor injection of G47? can induce a therapeutic effect and prolong the survival of treated mice bearing SMMC-7721 and BEL-7404 subcutaneously (s.c.) tumors. Thus, G47? may be useful as a novel therapeutic agent for HCC.