Objective：This study aims to establish Nudt3 knockout mouse model. Methods：According to the principle of CRISPR/Cas9-mediated gene editing technology，two gRNAs were separately designed at the exon 2 and the region between the exon 2 and the exon 3 of the Nudt3，and germline chimeric mice were constructed by microinjection of early embryos，from which suitable gene knockout mice were screened. RT-PCR and Western blot were used to verify the expression of Nudt3 at mRNA and protein levels. Results：We obtained gene editing mice of F0 generation with high efficiency，heterozygous mice of F1 generation and homozygous mice of F2 generation through breeding. The Western blot results showed that there was no expression of NUDT3 in several metabolic regulatory tissues of F2 generation homozygous mice. Conclusion：Nudt3-knockout mice are successfully constructed，this provides an ideal animal model for further metabolic phenotype analysis and mechanism study.