A major challenge for human allogeneic islet transplantation is the development of effective methods to induce donor-specific tolerance to obviate the need for life-long immunosuppression that is toxic to the insulin-producing beta cells and detrimental to the host. We developed an efficient donor-specific tolerance therapy that utilizes infusions of ethylene carbodiimide (ECDI)-treated donor splenic antigen-presenting cells that results in indefinite survival of allogeneic islet grafts in the absence of immunosuppression. Furthermore, we show that induction of tolerance is critically dependent on synergistic effects between an intact programmed death 1 receptor-programmed death ligand 1 signaling pathway and CD4(+)CD25(+)Foxp3(+) regulatory T cells. This highly efficient antigen-specific therapy with a complete avoidance of immunosuppression has significant therapeutic potential in human islet cell transplantation.