In eukaryotic cells, membranes of the late secretory pathway contain a disproportionally large amount of cholesterol in relation to the endoplasmic reticulum, nuclear envelope and mitochondria. At one extreme, enrichment of the plasma membrane with cholesterol and sphingolipids is crucial for formation of liquid ordered domains (rafts) involved in cell communication and transport. On the other hand, regulatory machinery in the endoplasmic reticulum is maintained in a relatively cholesterol-poor environment, to ensure appropriate rapid responses to fluctuations in cellular sterol levels. Thus, cholesterol homeostasis is absolutely dependent on its distribution along an intracellular gradient. It is apparent that this gradient is maintained by a combination of sterol-lipid interactions, vesicular transport and sterol-binding/transport proteins. Evidence for rapid, energy-independent transport between organelles has implicated transport proteins, in particular the eukaryotic oxysterol binding protein (OSBP) family. Since the founding member of this family was identified more than 25 years ago, accumulated evidence implicates the 12-member family of OSBP and OSBP-related proteins (ORPs) in sterol signalling and/or sterol transport functions. The OSBP/ORP gene family is characterized by a conserved β-barrel sterol-binding fold but is differentiated from other sterol-binding proteins by the presence of additional domains that target multiple organelle membranes. Here we will discuss the functional and structural characteristics of the mammalian OSBP/ORP family that support a ‘dual-targeting’ model for sterol transport between membranes.