Objective: To establish ACE2 (angiotensin converting enzyme 2, ACE2) gene-modified porcine fetal fibroblast (PFF) cell lines via CRISPR/Cas9 system-mediated gene modification, which could serve as donor cells for the subsequent construction of a SARS-CoV-2 insensitive pig model. Methods: Firstly, the ACE2 amino acid sequences of rats and chickens were compared by bioinformatic analysis. The ACE2 amino acid residues, which play a crucial role in the interaction between ACE2 and SARS-CoV-2 S protein, were replaced by the amino acid residues of ACE2 chickens at the corresponding sites. The chimeric ACE2 gene was synthesized and cloned into the knock-in vector. Secondly, sgRNA targeting the porcine ACE2 gene was designed, synthesized, and cloned into the pX330 vector to construct the ACE2 gene targeting vector. Finally, the ACE2 targeting vector, the ACE2 gene knock-in donor vector, and a neomycin-resistant plasmid were co-transfected into porcine fetal fibroblasts. G418 was used to screen the resistant single-cell colonies, and sequencing was performed to identify the correct ones. Results: According to the amino acid sequence alignment, the rat ACE2 amino acid residues at positions 19th, 31st, 34th, 35th, 42nd, 353rd, and 354th were replaced by the amino acid residues of chicken ACE2. The chimeric ACE2 gene was synthesized, and the ACE2 knock-in vector was successfully constructed. Genotype analysis of G418 resistant colonies showed that single-cell colonies with expected modification were successfully obtained. Conclusion: The PFFs with endogenous ACE2 knock-out and chimeric ACE2 knock-in were successfully generated using CRISPR/Cas9-mediated gene modification, which will lay a foundation for the construction of SARS-CoV-2 insusceptible pig models.