Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) is a cancer-specific, apoptosis-inducing gene with broad-spectrum antitumor activity, making it an ideal candidate for a novel cancer gene therapy. A systemic and sustained antitumor immune response generated at the time of initial molecular-targeted therapy would provide additional clinical benefits in cancer patients, resulting in improved prevention of tumor recurrence. In this study, we explored the therapeutic efficacy of intratumoral delivery of a nonreplicating adenoviral vector encoding mda-7/IL-24 (Ad.mda-7) and a secretable form of endoplasmic reticulum resident chaperone grp170 (Ad.sgrp170), a potent immunostimulatory adjuvant and antigen carrier. Intratumoral administration of Ad.mda-7 in combination with Ad.sgrp170 was more effective in controlling growth of TRAMP-C2 prostate tumor compared with either Ad.mda-7 or Ad.sgrp170 treatment. Generation of systemic antitumor immunity was shown by enhanced protection against subsequent tumor challenge and improved control of distant tumors. The combined treatments enhanced antigen and tumor-specific T-cell response, as indicated by increased IFN-gamma production and cytolytic activity. Antibody depletion suggests that CD8(+) T cells may be involved in the antitumor effect of the dual molecule-targeted therapies. Therefore, introducing immunostimulatory chaperone grp170 in situ strongly promotes the "immunogenic" cell death when delivered to the mda-7/IL-24-induced apoptotic tumor cells, indicating that an improved anticancer efficacy may be achieved by concurrently targeting both tumor and immune compartments. Given multiple undefined antigens present endogenously within prostate cancer, these data provide a rationale for combining sgrp170-based vaccine strategy with mda-7/IL-24-targeted cancer therapy to induce durable systemic immunity.