B7-H1 is a co-inhibitory molecule belonging to the B7 family. The B7-H1 protein is only expressed on macrophage lineage of cells in normal tissues, but is overexpressed in most types of tumor. The aberrant expression of cell surface B7-H1 on cancer cells is generally associated with high-risk prognostic factors. The tumor-associated B7-H1 increases apoptosis of antigen-specific T cells through interaction with its receptor PD-1 on CD8+ T cells and contributes to tumor immune evasion. These features suggest that B7-H1 may be a therapeutic target for the B7-H1-expressing tumors. We developed a therapeutic vaccine by coupling a tetanus toxoid T-helper cell epitope with the N-terminal of B7-H1 IgV-like domain. This vaccine was able to induce high titers of antibodies against B7-H1 in mice which were able to bind to native cell surface B7-H1. We chose the B7-H1-expressing SP2/0 myeloma and its syngeneic host (the BALB/c mouse) as the model to study the antitumor activity of the rhB7-H1M vaccine. Vaccination with this modified B7-H1 protein resulted in almost complete protection from SP2/0 tumor challenge and efficiently eliminated pre-established tumors in mice. In addition, B7-H1 vaccination was able to decrease the percentage of CD4+ Foxp3+ regulatory T cells in tumor-bearing mice and which might improve antitumor immunity. These data demonstrate the potential of B7-H1-based vaccine as a therapeutic agent for the treatment of cancer overexpressing B7-H1.