The progression of cancer is largely determined by the interactions between particular lymphocytes and cancer cells. Regulatory T cells (Tregs) suppress self-reactive lymphocytes to prevent autoimmune disease. In cancer patients, Tregs serve a protumorigenic function, as their presence in the tumor microenvironment interferes with the ability of other lymphocytes to attack cancer cells. Thus, elevated levels of Tregs correspond to poor prognoses. Evidence shows that the removal of Tregs can enhance the anti-tumor immune response; in certain cancers, the depletion of Tregs is considered an effective cancer immunotherapy treatment strategy. Regular exercise has been reported to reduce the risk of cancer recurrence and mortality, as well as regulate and deter the growth of tumors. It is inferred that exercise indirectly impacts cancer by affecting specific lymphocytes. This research is concerned with the effect of exercise on regulatory T cell populations in the tumors and spleens of tumor-bearing rats. It was hypothesized that exercise decreases the proliferation of Treg cells in the tumor microenvironment and lymphoid tissue. For this study, tumor and spleen samples were collected from sedentary and exercised cohorts of rats with breast cancer. CD25 antibody, Foxp3 antibody, and Hoechst dye were applied to the tissues so that samples could be visualized using fluorescence microscopy to identify Tregs. Overall trends in the data suggest that exercise reduces Treg cell count in both the tumors and spleens of individuals, thereby limiting the extent to which Tregs can suppress the anti-tumor immune response in cancer patients. As this was the first successful Foxp3 IHC protocol to be used in rats, implementing a larger sample size, CD24 antibodies, confocal microscopy, and flow cytometry in future studies would enable stronger conclusions to be drawn in regards to the effect of exercise on Treg populations in cancer patients.