Current cancer gene therapies aim at the induction of systemic antitumor immune responses. Tumors may deliver antigens to T-cells, but may lack the costimulatory signals necessary for mounting an effective response. The purpose of this study was to evaluate the efficacy of an adenoviral delivery of the B7-H3 costimulatory molecule in mice to induce antitumor immune responses. Colon cancers were established by orthotopic injection of syngeneic colon cancer cells into the cecum on Balb/c mice. After two weeks, these mice were treated by intratumoral injection of an adenovirus expressing mouse B7-H3 (Ad-B7-H3-GFP) or a control virus (Ad-GFP). Ad-B7-H3-GFP treatment resulted in a reduction of tumor size compared to the controls. In addition, the occurrence of secondary metastasis was significantly reduced in B7-H3 treated mice compared to control animals (lymph node 7/10 vs. 10/10; liver 2/10 vs. 8/10, p<or=0.05). Ad-B7-H3-GFP treated animals showed significantly higher frequencies of tumor-specific interferon-gamma producing CD8+ T-cells (p<or=0.05) and higher interleukin-12 levels (p<or=0.01) than control animals. This study demonstrates that adenoviral B7-H3 transfer is able to induce a specific cellular antitumor immune response leading to primary tumor regression and reduction of secondary metastasis in vivo.