Biological membranes are composed of lipid bilayers. Major lipid components of the eukaryotic plasma membrane include glycerophospholipids, sphingolipids, and cholesterol. Lipids are irregularly distributed between the two leaflets, thus causing lipid asymmetry, or within the same leaflet, forming a lipid microdomain. Glycerophospholipids and sphingolipids both contribute to the lipid asymmetry, whereas cholesterol and sphingolipids form lipid microdomains. Maintenance of proper lipid asymmetry is required for the mechanical stability of the membrane and for vesicular transport. On the other hand, local or global changes in lipid asymmetry are important for cell cycle progression, apoptosis, and platelet coagulation. Three classes of lipid translocases, P-type ATPases, ABC transporters, and scramblases, are known to be involved in the regulation of lipid asymmetry. In this review, we describe the physiological and pathological functions of lipid asymmetry and the current knowledge of lipid translocases.