Proline accumulation is one of the most common reactions in plants under drought stress. Pyrroline-5-carboxylate reductase (P5CR) is the final enzyme and plays an important role in proline biosynthesis. The Chinese wild grapevine Vitis yeshanensis J.X. Chen accession 'Yanshan-1' is highly resistant to drought, but the genetic and molecular mechanisms associated with this resistance have not been elucidated. Here, we cloned a VyP5CR gene (Genbank ID: MZ226960) from 'Yanshan-1', and evaluated its transcriptional response to drought, NaCl, cold, as well as exogenous ABA, MeJA and SA. Tissue specific analysis showed that VyP5CR could be expressed in various organs and was highly expressed in roots. To gain insight into the roles of VyP5CR, we overexpressed VyP5CR in Arabidopsis thaliana (Arabidopsis). Transgenic Arabidopsis plants expressing VyP5CR showed enhanced survival rate, smaller stomata in response to severe drought, as well as stronger root growth on a medium containing mannitol. Under drought stress, VyP5CR-OE plants showed reduced levels of MDA, H₂O₂ and O₂⁻, and higher proline content, SOD and POD activity. In addition, VyP5CR-OE plants showed increased induction of the drought-related genes COR15A, COR47, DREB2A, KIN1, NCED3 and RD29A. Taken together, these experiments reveal that VyP5CR can promote the drought tolerance of transgenic Arabidopsis. Besides, an interacting protein with VyP5CR, VyCSN5B (COP9 signalosome complex subunit 5b), was screened out by yeast two-hybrid and verified by bimolecular fluorescence complementation assay.