The immune response due to Galα1,3-Galβ1-4GlcNAc-R(α-Gal) epitopes is an important factor in bioprosthetic heart valve failure. The aim of this study was to evaluate the immune reaction and anticalcification effect of α-galactosidase and decellularization for glutaraldehyde (GA)/genipin fixed bovine pericardium using α1,3-galactosyltransferase knockout(α-Gal KO) mouse(C57BL/6). Bovine pericardial tissues were decellularized and treated with α-galactosidase before fixation with 0.25% GA/0.4% genipin in organic solvent (75% ethanol and 5% octanol) and treatment with glycine. The removal of α-gal epitope from the bovine pericardium was analyzed by 3,3'-Diaminobenzidine staining intensity. The bovine pericardial tissues were subcutaneously implanted into wild type mice (n=19) and α-Gal KO mice (n=66), which had been presensitized with rabbit red blood cells to maximize immunologic response or not, and anti α-Gal antibodies were measured at various time intervals. Calcium contents of the explanted tissues (n=104) were measured 3 months after implantation. The treatment of α-galactosidase effectively removed the α-gal epitopes expressed on bovine pericardial tissues. In both GA and genipin groups, titers for both anti α-Gal IgM and IgG of α-Gal KO mice increased according to the duration of implantation, and were lower in the groups with decellularization than without decellularization, and were lower in the groups with α-galactosidase+decellularization than with decellularization. The calcium contents of GA/genipin fixed tissues were lower in the groups with decellularization than without decellularization, and were lower in the groups with α-galactosidase+decellularization than with decellularization. Treatment of α-galactosidase with decellularization is useful for removal of the immunogenicity, and reduced calcification in both GA and genipin fixed bovine pericardia, supporting the hypothesis that the immune reaction may cause the calcification. Treatment of α-galactosidase has possible promise to enhance durability of bioprosthetic heart valve. To our knowledge, this is the first report that demonstrates the in vivo efficacy of α-galactosidase using presensitized α-Gal KO mouse to mimic the human immunologic environment.