Pancreatic cancer is the fourth most deadly cancer in the United States. Despite development in conventional treatment strategies the 5 year survival rate is only 7.7%. In this study we demonstrated that the tripartite treatment by combination of fractionated radiation therapy, hyperthermia and anti-OX40 immunotherapy (tripartite) led to significant impact on pancreatic cancer in mice. The treatment of mice with the tripartite treatment demonstrated significant tumor growth inhibition (p<0.0001) with no observable toxicity due to this treatment. Flow cytometric analysis of the tumor showed a shift in tumor microenvironment from immune suppressive to immune stimulatory with significantly higher CD4+ and CD8a+ (p<0.05) T lymphocytes. A significantly higher population of helper T cells and cytotoxic T cells was observed in the usually immune-deficient pancreatic cancer tumor microenvironment coupled with a decrease in the immunosuppressive microenvironment in the tumors of animals receiving the tripartite treatment is potentially the cause of the superior anti-tumor effect observed in animals receiving the tripartite treatment.