To understand cold acclimation and cold tolerance in Vitis, we isolated a C-repeat binding factor 1 (CBF1) transcriptional activator from cold-sensitive Vitis vinifera ‘Manicure Finger’ and cold-tolerant wild Vitis amurensis. Under cold stress, the CBF1 transcript accumulation of V. amurensis increased, whereas that of V. vinifera showed no significant change. The transcript levels of VaCBF1 in the roots, stems, leaves, and petioles under cold stress were up-regulated in a time-dependent manner. The transcript level of VaCBF1 in the leaves was induced by salinity stress or by exogenous abscisic acid and salicylic acid. The presence of the cis-elements MBS, MYB, and MYC in the VaCBF1 promoter suggests that this promoter is a component of the CBF transduction pathway, which is involved in plant response to cold stress. The overexpression of VaCBF1 increased the cold tolerance of transgenic tobacco at −4 °C. The transcript level of the downstream target gene NtERD10D appeared in the transgenic lines under normal conditions, whereas that of NtERD10D and NtDREB3 improved under low temperature. We suggest that VaCBF1 enhances stress tolerance by increasing antioxidant activities and promoting downstream target gene expression.