Despite substantial data elucidating the roles of cholesterol in lipid rafts at the plasma membrane, the roles of cholesterol and related lipids in lipid raft microdomains at the level of subcellular membrane, such as the endoplasmic reticulum (ER) membrane, remain less understood. Growing evidence, however, begins to unveil the importance of cholesterol and lipids on the lipid raft at the ER membrane. A few ER proteins including the sigma-1 receptor chaperone were identified at lipid raft-like microdomains of the ER membrane. The sigma-1 receptor, which is highly expressed at a subdomain of ER membrane directly apposing mitochondria and known as the mitochondria-associated ER membrane or MAM, has been shown to associate with steroids as well as cholesterol. The sigma-1 receptor has been implicated in ER lipid metabolisms/transports, lipid raft reconstitution at the plasma membrane, trophic factor signalling, cellular differentiation, and cellular protection against beta-amyloid-induced neurotoxicity. Recent studies on sigma-1 receptor chaperones and other ER proteins clearly suggest that cholesterol, in concert with those ER proteins, may regulate several important functions of the ER including folding, degradation, compartmentalization, and segregation of ER proteins, and the biosynthesis of sphingolipids.