Abstract Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-beta (IFN-beta) gene (VSV.IFN-beta). We conducted this study to determine the ability of VSV.IFN-beta to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-beta did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-beta. In the eight resistant lines, pretreatment with IFN-beta prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-beta protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2',5'-oligo-adenylate-synthetase (2'5'-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-beta protein and no IFN- or VSV-induced changes in PKR, MxA, and 2'5'-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-beta by immunostaining for the presence of p48 protein.