Promoting immune tolerance to transplanted organs can minimize the amount of immunosuppressive drugs that patients need to take, reducing lifetime risks of mortality and morbidity. Regulatory T cells (T _regs ) are essential for immune tolerance, and preclinical studies have shown their therapeutic efficacy in inducing transplantation tolerance. Here, we report the results of a phase 1/2 trial (ARTEMIS, NCT02474199) of autologous donor alloantigen–reactive T _reg (darT _reg ) therapy in individuals 2 to 6 years after receiving a living donor liver transplant. The primary efficacy endpoint was calcineurin inhibitor dose reduction by 75% with stable liver function tests for at least 12 weeks. Among 10 individuals who initiated immunosuppression withdrawal, 1 experienced rejection before planned darT _reg infusion, 5 received darT _regs , and 4 were not infused because of failure to manufacture the minimal infusible dose of 100 × 10 ⁶ cells. darT _reg infusion was not associated with adverse events. Two darT _reg -infused participants reached the primary endpoint, but an insufficient number of recipients were treated for assessing the efficacy of darT _regs . Mechanistic studies revealed generalized T _reg activation, senescence, and selective reduction of donor reactivity after liver transplantation. Overall, the ARTEMIS trial features a design concept for evaluating the efficacy of T _reg therapy in transplantation. The mechanistic insight gained from the study may help guide the design of future trials.