Objective：This study aims to build inbred Wuzhishan miniature pigs with GGTA1/β4GalNT2 double gene knockout by CRISPR/Cas9 mediated targeting. Methods: Single-guide RNAs（sgRNAs） specific for the pig GGTA1 and β4GalNT2 were designed and synthesized，then cloned into the pX330 plasmid containing a Cas9 skeleton，respectively. The resulting targeting vectors for GGTA1 and β4GalNT2 were transfected into the primary porcine fetal fibroblasts（PFFs） derived from Wuzhishan miniature inbred pigs. G418 drug screening and Sanger sequencing were used to identify the monoclonal cells with GGTA1/β4GalNT2 double gene knockout. Somatic cell nuclear transfer（SCNT） was employed to generate Wuzhishan miniature inbred pigs using the obtained colonies as donor cells. Flow cytometry analysis was conducted to examine the αGal and Sd（a） antigen expression in peripheral blood mononuclear cell（PBMC） of the cloned piglets. Results：Cas9/sgRNA expression vectors targeting pig GGTA1 and β4GalNT2 genes were successfully constructed. After transfection into PFFs，9 cell colonies were obtained with biallelic modifications in both GGTA1 and β4GalNT2 loci. Ten cloned piglets were produced by SCNT. The expression of αGal and Sd（a）antigens on these cloned knockout piglets was negative. Conclusion：The CRISPR/Cas9 system showed high efficiency in pig gene targeting. The GGTA1/β4GalNT2 double gene knockout inbred Wuzhishan miniature pigs were successfully produced in the present study，which could serve as newly ideal materials for xenotransplantation.