The plasma membrane (PM) is one of the most interactive and dynamic membrane structures of the cell. It represents an interface between the cell and the extracellular environment and, as such, it is involved in many biological processes, e.g., metabolite and ion transport, endocytosis, defense against pathogens, cell differentiation and proliferation. The PM contains microdomains enriched in sphingolipids and sterols that are resistant to certain concentrations of detergents. The aim of this work was to determine the main functions of such microdomains in poplar cells through quantitative proteomics, using gel-based and solution (iTRAQ) approaches. Compared to PM, 80 proteins related to cell wall biosynthesis, transport, stress responses and signaling processes were found to be significantly enriched in the microdomains. The majority of these proteins were predicted to contain up to 16 transmembrane domains and/or membrane-anchoring acylation sites. The occurrence of S-acylation sites in PM and microdomain proteins was experimentally determined using an acyl-biotin exchange method and mass spectrometry. Out of the 450 S-acylated proteins in PM, 24 were enriched in microdomains, suggesting S-acylation plays a key role in the localization of these proteins to specialised PM subdomains. In addition, the number of transmembrane domains and their length were higher in the proteins isolated from the microdomains than in the total PM proteins. An important proportion of the most enriched microdomain proteins corresponded to cell wall biosynthetic enzymes, namely (1®3)-β-glucan (callose) synthases and related proteins, indicating that the isolated microdomains are the site for callose biosynthesis and regulation. The proteins identified most likely reflect the biological specialisation of the isolated microdomains in cell surface specific responses that trigger callose formation, e.g. stress responses, as well as a potential role in plasmodesmata formation and structure. The data will be discussed in relation with these key biological processes in plant cells.