The primary immunologic barrier to overcome before clinical xenotransplantation can be successful is rejection mediated by preformed natural antibodies in the host, directed toward a single carbohydrate epitope Galα1-3Galβ1-4GlcNAc-R (αGal) present on porcine tissue, encoded for by the enzyme glucosyltransferase UDP galactose:β-D-galactosyl-1,4-N-acetyl-D-glucosaminide α(1-3)galactosyltransferase (EC 2.4.1.151) or simply αGT. Although we have shown previously that a gene therapy approach could be used to prevent production of natural antibodies specific for αGal, the ability to induce and maintain tolerance after rigorous antigen challenge would be required if similar approaches are to be used clinically. Here, we demonstrate in αGT knockout mice (GT⁰ mice), which, like humans, contain in their serum antibodies that bind αGal, that the efficient transduction and expression of a retrovirally transduced αGT gene in bone marrow–derived cells induces stable long-term tolerance to the αGal epitope. GT⁰ mice reconstituted with αGT-transduced bone marrow cells were unable to produce antibodies that bind αGal after extensive immunization with pig cells. Furthermore, using ELISPOT assays, we were unable to detect the presence of B cells that produce αGal reactive antibodies after immunization, suggesting that such B cells were eliminated from the immunologic repertoire after gene therapy. Interestingly, after tolerance to αGal is induced by gene therapy, the antiporcine non-αGal humoral response changes from a predominantly IgM to an IgG response. This suggests that once the natural antibody barrier is eliminated by the induction of tolerance, the antipig response changes to a typical T-cell–dependent response involving isotype switching. Thus, gene therapy approaches may be used to overcome immunologic responses leading to xenograft rejection, and similar gene therapy approaches could be used to overcome autoimmunity.