The suppressive activity of regulatory T cells (Treg) has been implicated as an important factor limiting immune mediated destruction of tumor cells. However, not much is known about the presence and function of Treg within tumors. Here we show in a syngeneic murine glioma model a time-dependent accumulation of CD4+FoxP3+ Treg in brain tumors. Further analysis revealed a time-dependent upregulation of CD25, CTLA-4, GITR and CXCR4 on intratumoral CD4+FoxP3+ Treg during tumor growth. Moreover, freshly isolated intratumoral Treg were highly suppressive when tested directly ex vivo. Treatment with anti-CD25 monoclonal antibodies (mAbs) significantly reduced the number of these highly suppressive CD4+FoxP3+ cells within the growing tumor and provoked a CD4 and CD8 T cell dependent destruction of the glioma cells. Combining Treg depletion with administration of blocking CTLA-4 mAbs further boosted glioma-specific CD4+ and CD8+ effector T cells as well as antiglioma IgG2a antibody titers resulting in complete tumor eradication without any signs of autoimmunity. These data illustrate that intratumoral accumulation and activation of CD4+FoxP3+ Treg act as a dominant immune escape mechanism for gliomas and underline the importance of controlling tumor-infiltrating Treg in glioma immunotherapy.