Tumor-derived glucose-regulated protein 94 (GRP94/gp96) has shown great promise as a tumor vaccine. However, current protein-based approaches require the availability of large quantities of tumor tissue, which are often not possible. In addition, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of a combined GRP94/gp96-based genetic immunotherapy and radiation therapy strategy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding a modified, secretable form of GRP94 gene (AdsGRP94) was constructed and evaluated in various antitumor experiments. Lethally irradiated, virus-infected cells were used as vaccines. Adenoviral vectors were also injected directly into tumors in conjunction with tumor irradiation. Vaccination with lethally irradiated, AdsGRP94-infected 4T1 cells completely prevented subsequent tumor growth from challenge inoculations of as many as 107 cells per mouse. In established tumor models, vaccinations alone had minimal effect on local and metastatic tumor growth. However, when vaccination was combined with radiation therapy and i.t. AdsGRP94 injections, local tumor growth and pulmonary metastasis were markedly inhibited. In some cases, complete tumor regression was observed. In these cases, the mice were resistant to subsequent tumor challenge and remain tumor free up to 10 months after initial therapy. Our results indicate that combined AdsGRP94-based immunotherapy and radiation therapy may be a potentially effective strategy for cancer treatment.