To enhance further the safety and efficacy of oncolytic vaccinia virus, we have developed a new virus with targeted deletions of three viral genes encoding thymidine kinase and antiapoptotic/host range proteins SPI-1 and SPI-2 (vSPT). Infection of human and murine tumor cell lines yielded nearly equivalent or a log lower virus recovery in comparison to parental viruses. Viral infection activated multiple caspases in cancer cells but not in normal cells, suggesting infected cells may die via different pathways. In tumor-bearing mice, vSPT recovery from MC38 tumor was slightly reduced in comparison to two parental viruses. However, no virus was recovered from the brains and livers of mice injected with vSPT in contrast to control viruses. vSPT demonstrated significantly lower pathogenicity in nude mice. Systemic delivery of vSPT showed significant tumor inhibition in subcutaneous MC38 tumor, human ovarian A2780 and murine ovarian MOSEC carcinomatosis models; however, the tumor inhibition by vSPT was reduced compared with parental viruses. These results demonstrated that although deletion of these three viral genes further enhanced tumor selectivity, it also weakened the oncolytic potency. This study illustrates the complexity of creating a tumor-selective oncolytic virus by deleting multiple viral genes involved in multiple cellular pathways.