Objective：To construct G protein-coupled receptor associated sorting protein 2（GPRASP2）-modified procine fetal fibroblasts（PFFs） as donor cells for the generation of GPRASP2-disrupted Bama miniature（BM）pigs. Methods：Bioinformatics methods were applied to phylogenetic and homologue analysis of human/porcine GPRASP2，and the secondary structures of human/porcine GPRASP2 proteins were predicted. Two single-guide RNAs（sgRNAs），targeting the upstream/downstream of coding sequence of the porcine GPRASP2，were designed，synthesized and ligated to pX330 plasmid. The recombinant plasmids containing Cas9 backbone were transfected into PFFs. Viable cell colonies were obtained using G418 screening and subjected to genotyping via direct PCR-based sequencing. Results：The human and porcine GPRASP2 proteins are evolutionarily closer，highly homologous，and predicted to have the similar functional Arm2 domains via 2D and 3D structure modeling. CRISPR/Cas9-sgRNA expression vectors targeting porcine GPRASP2 were constructed and transfected into PFFs. GPRASP2-deficient monoclonal PFFs were obtained by drug screening，genotypic analysis and Western blot assay. Conclusion：The human/porcine GPRASP2 proteins are evolutionarily closer and highly homologous. The GPRASP2-deficient cells were successfully constructed via CRISPR/Cas9 mediated gene editing，which provided a substantial foundation for the generation of GPRASP2-disrupted BM pigs.