Microdomains in the plasma membrane (PM) have been proposed to be involved in many important cellular events in plant cells. To understand the role of PM microdomains in plant cold acclimation, we isolated the microdomains as detergent-resistant plasma membrane fractions (DRMs) from Arabidopsis seedlings and compared lipid and protein compositions before and after cold acclimation. The DRM was enriched in sterols and glucocerebrosides, and the proportion of free sterols in the DRM increased after cold acclimation. The protein-to-lipid ratio in the DRM was greater than that in the total PM fraction. The protein amount recovered in DRMs decreased gradually during cold acclimation. Cold acclimation further resulted in quantitative changes in DRM protein profiles. Subsequent mass spectrometry and Western blot analyses revealed that P-type H-ATPases, aquaporins and endocytosis-related proteins increased and, conversely, tubulins, actins and V-type H-ATPase subunits decreased in DRMs during cold acclimation. Functional categorization of cold-responsive proteins in DRMs suggests that plant PM microdomains function as platforms of membrane transport, membrane trafficking and cytoskeleton interaction. These comprehensive changes in microdomains may be associated with cold acclimation of Arabidopsis.