To understand the mechanism by which the barley plants tolerate overwintering, we analyzed the BLT101 gene expression in four different cultivars including two each of spring and winter barleys, where the BLT101 transcript level varied among the cultivars with a long-term cold acclimation. The BLT101 transcript was induced at a higher level in the crown tissues in response to low temperature treatment. The BLT101 transcript level correlated with those of the corresponding protein. BLT101-overexpressing transgenic wheat lines (BLT101ox’s) grew slowly and showed decreased expression of the genes induced by hormones (such as auxin and cytokinin), compared to non-transgenic (NT) plants. After a prolonged cold treatment, the BLT101ox leaves showed normal phenotypes, while the NT plants displayed blighted and withered leaves. A substances leakage test showed that the BLT101ox wheat retains enhanced freezing tolerance, compared to the NT plants. The transgenic lines lost less water than the NT plants, under a cold-stressed condition. Our findings suggest that tolerance to water stress and the retardation of growth in cold conditions, the essential features in overwintering barleys, involve BLT101.