Objective  To construct homozygous aquaporin 9(AQP-9)-/- mice using the CRISPR/Cas9 system.    Methods  According to the design principle of CRISPR/Cas9 target, the exon region of the AQP-9 gene sequence was found in the Ensembl database. AQP-9-202 exon was selected through comprehensive analysis. In the early stage, 7 small guide RNA(sgRNA) targets were designed on both sides of it, appropriate targets were selected and AQP-9 was knocked out. The knockout result  were detected by PCR and gene sequencing. After the F1 heterozygous mice were obtained, 10 wild-type mice (5 males and 5 females) were provided for breeding in order to obtain homozygous AQP-9-/- mice.    Results   After injection of 74 fertilized eggs and transplantation of 60 pieces, 6 F0 generation positive mice were obtained.After breeding and identification, the homozygous AQP-9-/- mice were finally obtained.    Conclusion  Homozygous AQP-9-/- mice with stable inheritance could be obtained by using the CRISPR/Cas9 system.