AGL16 is an important negative regulatory transcription factor that regulates stomatal density and ABA content in Arabidopsis, and plays an important role in drought resistance in Arabidopsis. In order to obtain Arabidopsis agl16 mutant material, we used cotton leaf crumple virus (CLCrV)mediated VIGE system to screen the target and knockout Arabidopsis AGL16 sgRNAs. At the same time, the intact editing vector was transformed into wildtype Arabidopsis by Agrobacteriummediated flower dip method, and Arabidopsis agl16 mutant was created and identified for drought resistance. The results showed that: (1) two sgRNAs that can target and knockout Arabidopsis AGL16 gene were obtained by CLCrVmediated VIGE system, while AtU626∷AtAGL16sgRNA135S∷Cas9Terp1300 editing vector was constructed to transform wildtype Arabidopsis Col0, and agl16 homozygous mutant (AGL16:4) with 4 bp deletion of the target site was screened. (2) The results of drought resistance phenotypic identification showed that after 18 days of drought treatment, most of the wildtype plants dried up and died, while the mutant plants had a relatively lightly stressed; After rehydration, the survival rates of wildtype and mutant plants were 34.5% (10/29) and 75% (27/36), respectively. (3) Compared with the wildtype, the number of stomata per unit area in leaves and the water loss rate of isolated leaves of AGL16:4 homozygous mutant plants were reduced under drought stress, but the seed amount per plant did not change significantly. The results showed that the drought resistance of AGL16:4 homozygous mutant was significantly enhanced compared with the wildtype, and the seed quantity was basically the same as that of the wildtype, indicating that AGL16 gene could be an ideal candidate gene for crop drought resistance breeding; The obtained agl16 mutant provides a favorable transgenic receptor material for the functional complementation verification of the AGL16 homologous gene cloned from crops in the future.