We established an orthotopic animal model of colon cancer in mice and applied this model to study the antitumor effects of B7-H3, the newest member of the B7 family of costimulatory molecules. Colon-26 murine colon adenocarcinoma cells were inoculated into the cecal subserosum of mice to induce colon tumor growth. The tumor growth rate and the survival time of the mice were observed. A stable B7-H3 transfected Colon-26 cell line was established and the immunogenic effect was investigated. All mice implanted with wild-type tumor cells had tumor growth in the colon and died. The mean survival rate was 23 days. Mice implanted with C26-B7-H3 had a significantly prolonged survival time of 38 days. Our data suggest that B7-H3 exerts an antitumor effect on adenocarcinoma of the colon and may be considered as an adjuvant immunotherapy in the treatment of colon cancers. Our orthotopic animal model of colon cancer in mice could be applied to in vivo experimental studies of colon cancer.