In order to study the function of miR473a in Populus euphratica, we cloned the precursor of miR473a, Pre-Peu-miR473a, and used floral dip method to transform miR473a into Arabidopsis thaliana. Using PCR and histochemical staining of β-glucuronidase (GUS) detection methods, we obtained the CaMV35S:miR473a transgenic plants. Then we validated the growth status and physiological indexes of transgenic plants and wild-type plants under hypertonic environment simulated by D-mannitol and natural soil drought conditions. The results indicated that the miR473a precursor has a length of 100 base pair, sharing 100% homology with miR473a homologue in Populus trichocarpa, and forming perfect secondary stem loop structure. Compared with wild-type A. thaliana, the transgenic plants exhibited higher germination rate, longer root length,and better growth conditions under osmotic stress (200 mmol/L D-mannitol); In soil drought condition, the plant height, relative water content (RWC) and maximum photosynthetic efficiency of transgenic plants were all significantly higher, 10%, than that of the wild type A. thaliana (P0.05). The semi-quantitative PCR results showed that Peu-miR473a gene is involved in up-regulation in P. euphratica under drought stress. The putative target genes in Populus Potri. 012G093900, Potri.007G100200, Potri.009G165300 and Potri.004G204400 are down-regulated by drought stress. Expressions of the potential target genes in A. thaliana AT1G24530, AT5G45000, AT5G46070 and AT3G52950 were all decreased in transgenic plants, indicating that they may be target-regulated by miR473a gene. Our study suggests that miR473a gene plays a role in plant drought tolerance by regulating the plants’ ability to resist dehydration and drought under stress.