Matching ABO blood group antigens between donors and recipients is critical to prevent hyperacute rejection in kidney transplantation. Enzymatic conversion of blood group antigens to the universal O type presents a promising strategy to overcome barriers in ABO-incompatible kidney transplantation. In this study, we employ α-galactosidase from Bacteroides fragilis to convert type B kidneys to type O during hypothermic machine perfusion. After 3 hours of perfusion with enzyme, more than 95% of blood group B antigens in the kidney endothelium are effectively removed. Subsequently, enzyme-treated kidneys are protected from antibody-mediated injuries in an ex vivo simulation of ABO-incompatible kidney transplantation. Encouraged by these results, a discarded type B kidney, following enzymatic conversion, is transplanted into a type O brain-dead recipient with high titer of anti-B antibody. The allograft survives for 63 hours without hyperacute rejection. Blood group B antigens re-express within 48 hours, with histopathological analyses indicating no evidence of antibody-mediated rejection. This enzymatic conversion approach holds the potential to broaden the practice of ABO-incompatible kidney transplantation, decrease waiting times and facilitate equitable organ allocation.