Abstract: The aim of this study was to establish IPEC-J2 cell lines with CD13 gene knockout by CRISPR/Cas9 genome editing technique and to reveal the role of cluster of differentiation 13 (CD13) in the porcine intestinal cells infected by pathogenic microorganisms. In this study, two guide RNAs (g3 and g5) were designed corresponding to the 2nd exon region of CD13 gene. The g3 and g5 were cloned into the pX330-GFP and pX330-RFP vector backbones, respectively. After 48 hours of transfection, cells with dual fluorescent labeling were sorted out by flow cytometry. Single cell clones were cultured, amplified by PCR, and then sequenced to obtain CD13 gene knockout positive cells. The results of PCR and sequencing of the obtained 20 cell clones showed that a total of 7 clones had fragment deletions, including one clone with a single allele fragment deletion, 3 clones with biallelic heterozygous fragment deletions, and 3 clones with biallelic homozygous fragments deletions. The protein expression levels of the biallelic homozygous fragment knockout cells were detected, and the results showed that CD13 protein was hardly expressed in the homozygous knockout cells, which indicated that IPEC-J2 cell lines with the CD13 knockout was successfully constructed. In this study, we obtained the CD13 knockout cell lines, which provided the foundation for studying the role of CD13 in the porcine intestinal cells infected by pathogenic microorganisms.