Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a nonpathogenic RNA virus with intrinsic oncolytic specificity due to attenuated antiviral responses in many tumors. We report that repeated hepatic arterial infusion of recombinant syncytia-forming VSV vector in advanced multifocal hepatocellular carcinoma (HCC)-bearing rats at a 10-fold reduced vector dose resulted in sustained tumor-selective virus replication until the onset of high-titer neutralizing antibodies in blood. No significant elevations in serum transaminases and liver pathology were noted, indicating a lack of hepatotoxicity. Substantially improved tumor response was achieved with completely necrotic tumor nodules surrounded by mononuclear phagocytic cells, followed by fibrosis and calcification of the lesions, angiogenesis, and regeneration of normal hepatic parenchyma. Survival of tumor-bearing rats treated with repeated vector infusions was not only significantly improved over that of animals after a single injection at 10 times the vector dose (P = .001), but 18% of animals in the former treatment group also achieved long-term and tumor-free survival compared with 0% of animals in the latter treatment group. In conclusion, this treatment regimen will be very useful in the future development of VSV-mediated virotherapy as a novel therapeutic modality for patients with advanced HCC. (HEPATOLOGY 2005;41:196–203.)