Cold stress is an important factor that limits grape (Vitis sp.) production around the world. The high expression of osmotically responsive genes 1 (HOS1) protein acts as a repressor of cold-responsive genes in plants. To increase understanding of mechanism regulating cold tolerance in grape, we isolated and characterized a novel HOS1 gene, designated VvHOS1 from ‘Muscat Hamburg’ grapevine (Vitis vinifera). Real-time polymerase chain reaction (PCR) analysis revealed that the expression of VvHOS1 could be induced by the application of exogenous abscisic acid and various abiotic environmental conditions such as low temperature, drought, and salinity. Moreover, VvHOS1 expression could also be induced by cold plus drought conditions (4 °C, 10% polyethylene glycol 6000). In addition, overexpression of VvHOS1 in arabidopsis (Arabidopsis thaliana) decreased the plants’ tolerance to cold, drought, and salt as well as negatively regulated the expression level of two stress-responsive genes, AtRD29A and AtCOR47. The results obtained in this study should help us to elucidate the function of VvHOS1 and understand the cold-responsive pathway in grapevine.