Regulatory T cells (T_regs) are key mediators of immune tolerance and feature prominently in cancer. Depletion of CD25⁺ FoxP3⁺ T_regs in vivo may promote T cell cancer immunosurveillance, but no strategy to do so in humans while preserving immunity and preventing autoimmunity has been validated. We evaluated the Food and Drug Administration–approved CD25-blocking monoclonal antibody daclizumab with regard to human T_reg survival and function. In vitro, daclizumab did not mediate antibody-dependent or complement-mediated cytotoxicity but rather resulted in the down-regulation of FoxP3 selectively among CD25^high CD45RA^neg T_regs. Moreover, daclizumab-treated CD45RA^neg T_regs lost suppressive function and regained the ability to produce interferon-γ, consistent with reprogramming. To understand the impact of daclizumab on T_regs in vivo, we performed a clinical trial of daclizumab in combination with an experimental cancer vaccine in patients with metastatic breast cancer. Daclizumab administration led to a marked and prolonged decrease in T_regs in patients. Robust CD8 and CD4 T cell priming and boosting to all vaccine antigens were observed in the absence of autoimmunity. We conclude that CD25 blockade depletes and selectively reprograms T_regs in concert with active immune therapy in cancer patients. These results suggest a mechanism to target cancer-associated T_regs while avoiding autoimmunity.