The immunogenic carbohydrates, known as xenoantigens such as the Galα1-3Gal (α-Gal) epitope, and the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), play a major role in the immune response to xenotransplantation. Optimal decellularization protocols and strategies beyond decellularization are essential to eliminate xenoimmunogenicity. The aim of this study was to evaluate the safety and efficasy of our novel decellularization protocol and carbohydrase such as Peptide N-Glycosidase F (PNGase-F) for the removal of xenoantigens. We investigated the biomechanical properties, and efficacy for xenoantigen removal through expression of carbohydrate-binding lectins in porcine pericardium decellularized and treated with PNGase-F. Our novel decellularization protocols using 0.25% SDS + Triton X-100 through the multi-step methods with hypotonic, isotonic and hypertonic buffer solution demonstrated complete decellularization without histological changes. There were no histological changes depending on PNGase-F treatment concentration (0~2 unit/ml). There were no significant differences in tensile stress, tensile displacement, tensile strain at break, and permeability tests among porcine pericardia treated with different concentrations of PNGase-F. PNGase-F-treated porcine pericardium was stained with Jacalin, MAL I, WGA, RCA, GSL, ECA, PNA, SBA, WFA, and DSL, and showed lower fluorescence than native pericardium. PNGase-F treatment also resulted in a significant inhibition of lectin binding levels in a concentration-dependent manner. The fluorescent signal of decellularization effectively decreased lectin expression. Additional PNGase-F treatment for decellularization significantly reduced lectin expression without differences in PNGase-F concentration, demonstrating the synergistic effect of decellularization and PNGase-F. In conclusion, our novel decellularization protocols combined with PNGase-F treatment effectively removed the carbohydrates-associated xenoimmunogenicity and maintained biomechanical stability for cardiac xenografts. Our strategies beyond decellularization for optimal tissue engineering of cardiac xenografts can improve the biocompatibility of the graft.