Abstract Radiotherapy (RT) enhances innate and adaptive anti-tumor immunity; however, the effects of RT on immune-suppressive regulatory T cells (Tregs), in the tumor microenvironment (TME) have not yet been fully elucidated. Although previous reports suggest a post-RT increase in Tregs, whether these Tregs are functionally suppressive has not been determined. To test the hypothesis that RT enhances the suppressive function of Tregs in the TME, we selectively irradiated implanted tumors using the Small Animal Radiation Research Platform (SARRP), which models stereotactic RT in human patients– followed by flow-cytometric and functional analyses of tumor-infiltrating lymphocytes (TIL). Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Tegs), and that these cells have higher expression of CTLA-4, 4-1BB, and Helios, consistent with activated/suppressive phenotype. This observation held true across several tumor models (B16/F10, RENCA, MC38). Notably, we found that post-RT, TIL-Tregs had equal or improved suppressive capacity compared with non-irradiated tumors. Our data also indicated that Tregs proliferate post-RT, more robustly than the other T cell subsets in the TME. In addition, the post-RT Treg expansion occurred when T cell migration was inhibited by Fingolimod - suggesting that the post-RT Treg increase is likely due to preferential proliferation of intratumoral Tregs. Our data also suggest that post-RT Treg expansion is independent of TGF-β and IL-33. Collectively, these data demonstrate that RT increases the phenotypically and functionally suppressive Tregs in the TME, and provide a rationale for treatment regimens that combine RT with Treg-targeting agents to maximize anti-tumor efficacy.