Osh4 is an oxysterol-binding protein homologue found in yeast that is essential for the intracellular transport of sterols and cell life. In this study, molecular dynamics simulations were used to investigate the binding of ergosterol, 25-hydroxycholesterol, and lipid moieties to Osh4. The binding energies between both sterols and Osh4 were dominated by van der Waals interactions with residues within the sterol binding pocket, and were further stabilized by water-mediated interactions with polar residues at the bottom of the binding pocket (W46, Q96, Y97, N165, Q181). Only Q96 was able to form direct hydrogen bonds with each sterol. Possible lipid binding sites on the surface of Osh4 were identified by docking four lipid moieties modeled from different lipid head groups (phosphatidylcholine, phosphatidylserine, phosphatidylinositol(4,5)biphosphate, and phosphatidylinositol(3,4,5)triphosphate). Though lipids docked to several regions along the protein surface, the most commonly encountered regions were found along the beta-barrel region, a loop formed by residues 236-244, and the mouth of the sterol binding pocket. Several residues identified in these regions, such as K168, A169, K173, and E412, are either included or adjacent to residues experimentally implicated in Osh4 membrane binding. Lipid docking did not result in favorable binding regions on the alpha(7) helix or the distal binding surface, suggesting that protein backbone conformational changes are needed for membrane attachment in these regions. Ultimately, understanding how Osh4 attaches to cellular membranes and binds sterol will lead to a clear understanding of how this protein transports sterols between organelles in vivo.