One of the most important problems of strawberry cultivation in temperate regions is winter cold injuries. Current study investigated impacts of foliar application of glycine betaine (GB) at 0, 0.5, 1, 2 and 4 mM concentrations on the cold hardiness of strawberry. The plants were divided into two groups: one group for evaluation of cold hardiness at temperatures of -6, -9, -12, -15 and -18 °C; and the other for study of some biochemical characteristics. Results showed that GB treatment increased soluble carbohydrate and proline concentrations in both leaf and crown tissues, total protein concentration in leaf, and relative water content in leaf as compared to those in control. Based on LT50 calculated from electrolyte leakage and tetrazolium staining test, the GB application increased cold hardiness in strawberry plant based on its concentration. The highest cold hardiness was found in the 2 mM GB concentration based on electrolyte leakage and tetrazolium staining tests at -13.3 and -15.3 °C. Meanwhile, the lowest values of cold hardiness were observed in the control treatments based on electrolyte leakage and tetrazolium staining tests at -10.2 and -11.0 °C. Significant correlations were found between soluble carbohydrate and proline concentrations in leaf and crown, and total protein concentration with LT50 calculated from electrolyte leakage and tetrazolium staining test. We conclude that application of 2 mM GB has the capacity to increase the freezing tolerance of strawberry and could be used as a prophylactic tool to reduce winter cold injury.