Bioprosthetic heart valves (BHVs) originating from pigs are extensively used for heart valve replacement in clinics. However, recipient immune responses associated with chronic calcification lead to structural valve deterioration (SVD) of BHVs. Two well-characterized epitopes on porcine BHVs have been implicated in SVD, including galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) whose synthesis are catalyzed by α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Ac hydroxylase (encoded by the CMAH gene), respectively. It has been reported that BHV from αGal-knockout pigs are associated with a significantly reduced immune response by human serum. Moreover, valves from αGal/Neu5Gc-deficient pigs could further reduce human IgM/IgG binding when compared to BHV from αGal-knockout pigs. Recently, another swine xenoantigen, Sd(a), produced by β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2), has been identified. To explore whether tissue from GGTA1, CMAH, and β4GalNT2 triple gene-knockout (TKO) pigs would further minimize human antibody binding to porcine pericardium, TKO pigs were successfully produced by CRISPR/Cas9 mediated gene targeting. Our results showed that the expression of αGal, Neu5G and Sd(a) on TKO pigs was negative, and that human IgG/IgM binding to pericardium was minimal. Moreover, the analysis of collagen composition and physical characteristics of porcine pericardium from the TKO pigs indicated that elimination of the three xenoantigens had no significant impact on the physical proprieties of porcine pericardium. Our results demonstrated that TKO pigs would be an ideal source of BHVs.Surgical heart valve replacement is an established lifesaving treatment for diseased heart valve. Bioprosthetic heart valves (BHVs) made from glutaraldehyde-fixed porcine or bovine tissues are widely used in clinics but exhibit age-dependent structural valve degeneration (SVD) which is associated with the immune response against BHVs. Three major xenoantigens present on commercial BHVs, Galactosea α1,3 galactose (αGal), N-glycolylneuraminic acid (Neu5Gc) and glycan products of β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2) are eliminated through CRISPR/Cas9 mediated gene targeting in the present study. The genetically modified porcine pericardium showed reduced immunogenicity but comparable collagen composition and physical characteristics of the pericardium from wild-type pigs. Our data suggested that BHVs from TKO pigs is a promising alternative for currently available BHVs from wild-type pigs.