In the yeast Saccharomyces cerevisiae, the molecular chaperone Hsp26 is one component of the heat-shock response. Hsp26 has the remarkable ability to directly sense increases in temperature and switch from an inactive state to a chaperone-active state. In this study, we report a functional analysis of Hsp26 in Arabidopsis thaliana and its response to freezing stress. After freezing stress, the HSP26 transgenic plants exhibited stronger growth than the wild-type plants. We found that over-expression of HSP26 in Arabidopsis increased the amounts of free proline and soluble sugars, elevated the expression of stress defense genes, and enhanced Arabidopsis tolerance to freezing stress. Taken together, our results indicate that Hsp26 may play an important role in the response of transgenic Arabidopsis plants to freezing stresses.