Purpose Antibody-mediated xenograft rejection is inhibited by knock-out (KO) of porcine carbohydrates (CHO), including Galα1,3Gal. The β4Gal epitope was discovered using serum from xenograft-sensitized baboons. However, it remains controversial if humans and baboons have detectable titers of preformed anti-β4Gal antibody (as humans do for Neu5Gc), and whether anti-β4Gal antibody modulates lung xenograft injury. Methods In serum from 14 baboons, pre-lung transplant and post-op day1 anti-pig Ab binding against GalTKO versus GalTKO.β4GalKO (double KO (DKO)) porcine aortic endothelial cells (PAECs) was measured in animals that had received a lung from a GalTKO pig and compared to those in which the donor animal had β4GalKO (or knock down), with (TKO) or without (DKO) additional Neu5GcKO. To evaluate Ab binding to pig tissue, lung biopsies from wildtype, GalTKO, DKO and TKO pigs after 30min of ex vivo xeno-perfusion with human blood were stained for IgG and IgM binding. In one case Ab binding to GalTKO and DKO cells was also measured at 1 and 31d in a long-surviving xenolung recipient. Results IgM Ab binding in naive baboons was consistently lower on GalTKO.β4GalKO relative to GalTKO PAECs. Decline in anti-non-Gal Ab levels on post-op d1 in vivo was consistently less using GalTKO.β4GalKO lungs (31%±16%, n=3) when compared to single KO lungs (58%±16%, n=7). In a long-surviving LTX recipient animal, sensitized anti-non Gal antibody binding was almost completely abrogated when using TKO cells. Lungs with multiple CHO KOs appear to have decreased IgM tissue deposition relative to GalTKO lungs after perfusion with human blood when analyzed by Immunohistochemistry. Conclusion β4Gal is a dominant antigen after lung xenotransplantation. Binding of preformed antibody (IgM>IgG) in humans and baboons is reduced by β4GalKO. Since the β4Gal epitope presumably contributes to pig lung xenograft injury, including the β4GalKO modification in donor pigs will likely help to better control Ab-mediated xenorejection pathways and advance xenotransplantation of the lung and other organs towards the clinic.