Tumor-bearing individuals have been reported to harbor increased numbers of Foxp3⁺ regulatory T cells (Treg), which prevent the development of efficient antitumor immune responses. Thus, Treg depletion has already been tested as a promising therapeutic approach in various animal models and entered clinical trials. However, the use of nonspecific Treg targeting agents such as CD25 depleting antibodies, which in addition to CD25⁺ Tregs also deplete recently activated CD25⁺ effector T cells, potentially masked the tremendous potential of this therapeutic strategy. To avoid such nonspecific effects, we used transgenic DEREG (depletion of regulatory T cells) mice, which express a diphtheria toxin receptor under control of the Foxp3 locus, allowing selective depletion of Foxp3⁺ Tregs even during ongoing immune responses. We showed that Foxp3⁺ Treg depletion induced partial regression of established ovalbumin (OVA)-expressing B16 melanoma, which was associated with an increased intratumoral accumulation of activated CD8⁺ cytotoxic T cells. The antitumor effect could be significantly enhanced when Treg depletion was combined with vaccination against OVA. To further assess whether this therapeutic approach would break self-tolerance, we crossed DEREG mice with RipOVA^low mice, expressing OVA as neo–self-antigen under control of the rat insulin promoter. In these mice, combined Treg depletion and vaccination also induced tumor regression without the onset of diabetes. Together, our data suggest that selective Treg targeting strategies combined with vaccinations against tumor-associated (self) antigens have the potential to evoke efficient antitumor responses without inducing overt autoimmunity. These findings might have implications for future therapeutic interventions in cancer patients. Cancer Res; 70(20); 7788–99. ©2010 AACR.