F-box proteins, widely existing in eukaryotes, were involved in cell cycle regulation, apoptosis and multiple hormone signal transduction. Recently, it was showed that F-box proteins mediated the responses to abiotic and biotic stresses, which is crucial to maintain the normal growth and development for plant. Drought and other abiotic stresses seriously affected plant growth and development. It is urgent to solve the problem which enhances the drought resistance of plant at present. The Grapevine, one of important fruit trees, was widely cultivated among China and world. Drought stress severely affected the plant growth and dramatically reduced the fruit quality of grapevine. Therefore, it has a great meaning to isolate the drought-responsive genes and analyze its functions for stress-resistant improvement in grapevine. According to the analysis of drought treated grapevine transcriptome, we isolated 11 F-box genes obviously up-regulated under drought stress. Pfam software analysis showed these F-box genes encode amino acid sequences having a complete F-box domain. Among them, VvF-box5 exhibited higher expression level compared with other genes and VvF-box5 is located on the 19th chromosome. There are 1 824 bp in opening reading frame of the VvF-box5, including 5 exons and 4 introns according to gene sequence analysis. VvF-box5 protein contains 1 F-box domain in its N terminal and a fibrinogen-like domain（FBD） and two leucine-rich repeat（LRR）in its C terminal. Protein secondary structure prediction analysis showed that the VvF-box5 contain 15 α-helixs and 14 β-pleated sheets. The promoter element analysis showed that the promoter of VvF-box5 contains multiple stresses-responsive elements, such as GA response element GARE-motif, MeJA response element CGTCA-motif, Drought stress related components ethylene-responsive element (ERE), heat stress-responsive element (HSE) and low temperature responsive element (LTR), light response cis elements ACE, Box 4 and Sp1 and other element related to cell cycle regulation and development. Real-time qRT-PCR showed that VvF-box5 responded to drought, salt, ABA and JA. Subcellular localization showed that VvF-box5 was mainly located in nucleus in onion epidermis cells. Overexression of VvF-box5 clearly improved the drought tolerance in transgenic plants. In addition, the fusion protein 6×His-VvF-box5 was induced and expressed in prokaryotic expression system and purified by Ni-NTA Resin, laying foundation for studying VvF-box5 functions.