Methionine residues of proteins are a major target for oxidation by reactive oxygen species (ROS), which are generated in response to a variety of stress conditions. Methionine sulfoxide (MetO) reductases are present in most organisms and play protective roles in the cellular response to oxidative stress, reducing oxidized MetO back to Met. Previously, an Arabidopsis MetO reductase, MsrB3, was identified as a cold-responsive protein. Here we report that MsrB3 functions in the process of cold acclimation, thus contributing to cold tolerance. In contrast to normal, wild-type plants, msrb3 mutant plants lost the ability to become tolerant to freezing temperatures following cold pre-treatment. Furthermore, when exposed to low temperature, msrb3 plants exhibited a larger increase in MetO and H(2)O(2) content and electrolyte leakage compared with wild-type and MsrB3 transgenic plants. It is also shown that MsrB3 is localized at the endoplasmic reticulum (ER). We propose that MsrB3 plays an important role in cold tolerance by eliminating MetO and ROS that accumulate at the ER during cold acclimation.