Grapevine growing areas are increasingly affected by drought, which has greatly limited global wine production and quality.  DEAD-box is one of the largest subfamilies of the RNA helicase family, and its members play key roles in the growth and development of plants and their stress responses.  Previous studies have shown the potential of DEAD-box genes in the drought stress responses of Arabidopsis and tomato, rice, and other crop species.  However, information about DEAD-box genes in grapevine remains limited.  In this report, a total of 40 DEAD-box genes were identified in grapevine and their protein sequence characteristics and gene structures were analyzed.  By comparing the expression profiles of VviDEADRHs in response to drought stress in different grapevine varieties, nine candidate genes (VviDEADRH10c, -13, -22, -25a, -25b, -33, -34, -36, and -39) were screened based on expression profiling data.  Combined with qRT-PCR results, VviDEADRH25a was selected for functional verification.  Heterologous overexpression of VviDEADRH25a in Arabidopsis showed the transgenic plants were more sensitive to drought stress than the control.  Both electrolyte permeability and malondialdehyde content were significantly increased in transgenic plants, whereas the chlorophyll content and superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) enzyme activities were significantly decreased.  Furthermore, VviDEADRH25a-overexpressing plants showed down-regulated expression levels of several drought stress-related marker genes, namely AtCOR15a, AtRD29A, AtERD15, and AtP5CS1, which indicated that they participated in the drought stress response.  In summary, this study provides new insights into the structure, evolution, and participation of DEAD-box RNA helicase genes in the response to drought stress in grapevines.