Transporting substances such as gases, nutrients, waste, and cells is the primary function of blood vessels. Vascular cells use membrane proteins to perform crucial endothelial functions, including molecular transport, immune cell infiltration, and angiogenesis.A thorough understanding of these membrane receptors from a clinical perspective is warranted to gain insights into the pathogenesis of vascular diseases and to develop effective methods for drug delivery through the vascular endothelium. This review summarizes stateof-the-art single-molecule imaging techniques, such as super-resolution microscopy, singlemolecule tracking, and protein–protein interaction analysis, for observing and studying membrane proteins. Furthermore, recent single-molecule studies of membrane proteins such as cadherins, integrins, caveolins, transferrin receptors, vesicle-associated protein-1, and vascular endothelial growth factor receptor are discussed.