Regulatory T cells (T_regs) are essential to prevent autoimmunity, but excessive T_reg function contributes to cancer progression by inhibiting antitumor immune responses. T_regs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor–β1 (TGF-β1). On the T_reg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human T_regs. These antibodies recognize a conformational epitope that requires amino acids GARP_137–139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by T_regs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human T_regs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.