Objective To use CRISPR/ Cas9 gene editing technology to efficiently construct Bpi gene-knockout mouse models and continue to breed, identify and establish stable genetic Bpi gene-knockout mouse strains. Methods Knockout targets were designed at the two ends of exons 2-3 of the Bpi gene in C57BL/ 6 mice, and high activity guide RNA ( gRNA) targets were selected. The small guide RNA(sgRNA)was transcribed in vitro and microinjected into fertilized eggs of C57BL/ 6 mice together with Cas9 mRNA. F0 mice were obtained after embryo transfer into the surrogate mice. Positive mice with gene mutation was confirmed via genotype identification and DNA sequencing. Results Microinjection with Cas9 mRNA yielded highly active sgRNA and 64 fertilized eggs in good condition, which were successfully transplanted into 2 surrogate mice, yielding 22 F0 mice. After PCR identification and DNA sequencing, a mouse that was positive for a 708 bp single- strand deletion was selected to be propagated. The deletion was detected in the F1 and F2 generations, and homozygous mice were obtained in the F2 generation. Conclusions Bpi-knockout mouse models were successfully constructed, laying a foundation for further study of the biological functions of the Bpi gene and its expression products.