Cholesterol is a ubiquitous molecule in biological systems, and in particular plays various important roles in mammalian cellular processes. The presence of cholesterol is integral to the structure and behavior of biological membranes, and profoundly influences membrane involvement in cellular mechanisms. This review focuses on the incorporation of cholesterol into synthetic nanomaterials and assemblies, focusing on LC phase behavior, morphology/self-organization and hydrophobic interactions, all important factors in the design of nanomedicines. We highlight cholesteryl conjugates, liposomes and polymeric micelles, focusing on self-assembly capabilities, drug encapsulation and intracellular delivery. An area of considerable interest identified in this review is the use of cholesteryl-functional vectors to deliver drugs or nucleic acids. Such applications depend on the ability of the nanoparticle carrier to associate with both the cellular and endosomal membrane.