Objective：To build OSBPL2-knockout porcine fetal fibroblasts（PFFs）based on CRISPR/Cas9 technology and lay the important preparative foundation for establishing an experimental animal model of deafness in miniature pigs with OSBPL2 defects. Methods：Bioinformatics methods were applied to analyze the collinearity and homology of OSBPL2 between human and pig. The secondary and tertiary structures of OSBPL2 protein of human and pig were predicted and simulated. Two single-guide RNAs（sgRNAs）respectively targeting the fifth and the sixth exon of pig OSBPL2 were designed，synthesized and cloned into pX330 plasmid. G418 was used to obtain the positive monoclonal cells after transfection into PFFs. Finally，the efficiency of CRISPR/Cas9 mediated knockout was assessed by the T7EN1 enzyme digestion assay and the genotypes of monoclonal cells were identified by sequencing analysis. Results：Bioinformatic analysis revealed that the OSBPL2 of human and pig had a good collinearity on chromosome，highly homologous amino acid sequence（88%）and similar functional domain characteristics. CRISPR/Cas9 expression vectors targeting OSBPL2 were constructed and transfected into PFFs. OSBPL2-knockout monoclonal cells with mutant genotypes were obtained by drug screening and confirmed by DNA sequencing. Conclusion：The human and pig OSBPL2 sequences and their protein structures are highly homologous. CRISPR/Cas9 expression vectors were constructed to achieve OSBPL2 gene targeting in PFFs. OSBPL2-knockout monoclonal cells were obtained，which could contribute to the construction of OSBPL2-knockout miniature pig model.