Grapevines, bearing fruit containing large amounts of bioactive metabolites that offer health benefits, are widely cultivated around the world. However, the cold damage incurred when grown outside in extremely low temperatures during the overwintering stage limits the expansion of production. Although the morphological, biochemical, and molecular levels in different Vitis species exposed to different temperatures have been investigated, differential expression of proteins in roots is still limited. Here, the roots of cold-resistant (Vitis. riparia × V . labrusca, T1) and cold-sensitive varieties (Cabernet Sauvignon, T3) at −4 ◦C, and also at −15 ◦C for the former (T2), were measured by iTRAQ-based proteomic analysis. Expression levels of genes encoding candidate proteins were validated by qRT-PCR, and the root activities during different treatments were determined using a triphenyl tetrazolium chloride method. The results show that the root activity of the cold-resistant variety was greater than that of the cold-sensitive variety , and it declined with the decrease in temperature. A total of 25 proteins were differentially co-expressed in T2 vs. T1 and T1 vs. T3, and these proteins were involved in stress response, bio-signaling, metabolism, energy , and translation. The relative expression levels of the 13 selected genes were consistent with their fold-change values of proteins. The signature translation patterns for the roots during spatio-temporal treatments of different varieties at different temperatures provide insight into the differential mechanisms of cold resistance of grapevine.