Adoptive cell transfer (ACT) immunotherapy administered with prior host immunosuppression significantly improved the anti-tumor efficacy in a murine model. However, bulk transfer of lymphocytes containing suppressor lymphocyte subsets, including regulatory T cells to mice bearing late-stage tumors impaired this anti-tumor effect. In this study, we investigated the enhanced anti-tumor efficacy by adoptive transfer of Treg-depleted autologous tumor infiltrating lymphocytes in advanced murine breast cancer. We found that, compared to bulk cell transfer, Treg-depleted cell transfer enhanced the activation and proliferation of both CD4(+) and CD8(+) T cells. Most importantly, the immune response deviated towards the Th1 response reflected by increased IFNgamma and reduced IL-4 secretion in both CD4(+) and CD8(+) T cells and an enhanced granzyme B release of CTL. Furthermore, the elicited Th1 response subsequently resulted in delayed tumor growth and prolonged mice survival as well as reduced lung metastasis in tumor-bearing nude mice. These results strongly indicated that Treg-depleted autologous cell transfer greatly enhanced Th1 type immune response, consequently leading to delayed tumor growth and reduced tumor burden. Therefore, ACT immunotherapy based on ex vivo selection of tumor-reactive lymphocytes resulted in enhanced anti-tumor immunity and provides important implications for further human studies.