Malignant pleural mesothelioma (MPM) is a fatal disease with a median survival of less than 14 months. For the first time, a genetically engineered vaccinia virus is shown to produce efficient infection, replication, and oncolytic effect against MPM. GLV-1h68 is a replication-competent engineered vaccinia virus carrying transgenes encoding Renilla luciferase, green fluorescent protein (both inserted at the F14.5L locus), beta-galactosidase (inserted at the J2R locus, which encodes thymidine kinase), and beta-glucuronidase (at the A56R locus, which encodes hemagglutinin). This virus was tested in six human MPM cell lines (MSTO-211H, VAMT, JMN, H-2373, H-2452, and H-2052). GLV-1h68 successfully infected all cell lines. For the most sensitive line, MSTO-211H, expression of green fluorescent protein (GFP) started within 4 hr with increasing intensity over time until nearly 100% of cells expressed GFP at 24 hr. All cell lines were sensitive to killing by GLV-1h68, with the degree of sensitivity predictable by infectivity assay. Even the most resistant cell line exhibited 44 +/- 3.8% cell survival by day 7 when infected at a multiplicity of infection of 1.0. Viral proliferation assays demonstrated 2-to 4-fold logarithmic replication of GLV-1h68 in the cell lines tested. In an orthotopic model, GLV-1h68 effectively prevented development of cachexia and tumor-related morbidity, reduced tumor burden, and cured MPM in both early and late treatment groups. GLV-1h68 was successfully used to treat MPM in vitro and in an orthotopic model (in vivo). These promising results warrant clinical investigation of GLV-1h68 as a novel agent in the treatment of MPM.