Objective: We generated anti-mouse C4d single chain variable fragment (scFv) using phage display and constructed anti-C4d CAR that consisted of extracellular anti-C4d scFv and intracellular CD28 and CD3ζ. Anti-C4d CAR Tregs were prepared by retroviral transduction of CAR into sorted CD62L+CD4+CD25+ Tregs. Anti-C4d CAR Tregs were expanded by stimulation of anti-CD3/CD28 beads with rapamycin and IL-2. As a ABOi ABMR model, hearts from human blood group A-transgenic BALB/c mice were transplanted into C57BL/6J mice sensitized by A antigens. CD45.1+ non-transduced, control CAR, or anti-C4d CAR Tregs were transferred into recipients. Methods: Anti-C4d CAR Tregs expressed Foxp3, CD25, CTLA-4, LAP, and GITR to similar extent as non-transduced Tregs. Anti-C4d CAR Tregs specifically bound to C4d, were activated by binding to C4d, and their suppressive activity against in vitro T cell proliferation was similar as non-transduced Tregs. Next, adoptive transfer of anti-C4d CAR Tregs significantly prolonged mouse ABOi heart allograft survival than PBS control (P < 0.05), whereas non-transduced or control CAR Tregs did not. Antibody-mediated histologic injury and expression of IFN-γ and TNF-α in heart allografts were attenuated in the anti-C4d CAR Treg group. Furthermore, infiltration of CD45.1+ Tregs around C4d+ endothelial cells in the anti-C4d CAR Treg group was more prominent than that in other groups. Results: Anti-C4d CAR Tregs improved ABOi heart allograft survival by suppressing ABMR and are a promising therapeutic agent for controlling ABMR as well as ABOi allograft rejection. Conclusions: Objective: Antibody-mediated rejection (ABMR) is the main hurdle in ABO blood group-incompatible (ABOi) transplantation. Recently, chimeric antigen receptor regulatory T cells (CAR Tregs) have been developed to improve antigen specificity, viability, and suppressive activity of Tregs. Interestingly, deposition of complement component 4d (C4d) is a marker of ABMR and is also found in most ABOi allograft tissues. Based on this finding, we developed anti-C4d CAR Tregs to suppress ABOi allograft rejection.   Methods: We generated anti-mouse C4d single chain variable fragment (scFv) using phage display and constructed anti-C4d CAR that consisted of extracellular anti-C4d scFv and intracellular CD28 and CD3ζ. Anti-C4d CAR Tregs were prepared by retroviral transduction of CAR into sorted CD62L+CD4+CD25+ Tregs. Anti-C4d CAR Tregs were expanded by stimulation of anti-CD3/CD28 beads with rapamycin and IL-2. As a ABOi ABMR model, hearts from human blood group A-transgenic BALB/c mice were transplanted into C57BL/6J mice sensitized by A antigens. CD45.1+ non-transduced, control CAR, or anti-C4d CAR Tregs were transferred into recipients. Results: Anti-C4d CAR Tregs expressed Foxp3, CD25, CTLA-4, LAP, and GITR to similar extent as non-transduced Tregs. Anti-C4d CAR Tregs specifically bound to C4d, were activated by binding to C4d, and their suppressive activity against in vitro T cell proliferation was similar as non-transduced Tregs. Next, adoptive transfer of anti-C4d CAR Tregs significantly prolonged mouse ABOi heart allograft survival than PBS control (P < 0.05), whereas non-transduced or control CAR Tregs did not. Antibody-mediated histologic injury and expression of IFN-γ and TNF-α in heart allografts were attenuated in the anti-C4d CAR Treg group. Furthermore, infiltration of CD45.1+ Tregs around C4d+ endothelial cells in the anti-C4d CAR Treg group was more prominent than that in other groups. Conclusions: Anti-C4d CAR Tregs improved ABOi heart allograft survival by suppressing ABMR and are a promising therapeutic agent for controlling ABMR as well as ABOi allograft rejection.