We previously identified the mouse RTVP-1 (mRTVP-1; related to testes-specific, vespid, and pathogenesis proteins) gene as a direct target of p53 with proapoptotic activities in various cancer cell lines, including prostate cancer. To test the therapeutic potential of mRTVP-1 we constructed an adenoviral vector capable of efficient transduction and expression of mRTVP-1 (AdmRTVP-1) and used this vector in an orthotopic, metastatic mouse model of prostate cancer. A single intratumoral administration of AdmRTVP-1 gene therapy significantly reduced primary tumor wet weight compared with control Adbetagal-injected tumors at two time points after injection with two different vector doses (p < or = 0.01 at 7 and 14 days). Spontaneous metastasis to lung was also significantly reduced (p < or = 0.02). Evaluation of treated tumors revealed increased apoptosis and lower microvessel density counts. In a rat aortic ring sprouting assay, AdmRTVP-1 inhibited endothelial cell sprouting compared with Adbetagal, confirming its antiangiogenic activity. These therapeutic activities were associated with a significant increase in survival from 22.9 to 26.8 days (p = 0.003) in this aggressive model of prostate cancer. Interestingly, there were significant increases in the infiltration of tumor-associated macrophages, dendritic cells, and CD8+ T cells, which persisted at 14 days posttreatment in the AdmRTVP-1-treated tumors compared with Adbetagal control-treated tumors. In addition, significantly increased natural killer and cytotoxic T lymphocyte activities were demonstrated in the mice with AdmRTVP-1-treated tumors. The unique therapeutic properties of AdmRTVP-1 gene therapy demonstrated in this study provide new opportunities for gene and immunotherapy of prostate cancer and potentially other malignancies.