Abstract The important role of cholesterol in human body is well known. Therefore its properties have been investigated in many aspects. However, still some of its surface physicochemical properties are not well understood. Among them are wetting and electrokinetic properties. Therefore the aim of this paper was to study these properties and to compare them with the published literature data. For this purpose the advancing and receding contact angles of water, formamide and diiodomethane on the surface of cholesterol pellets and on its layers deposited on glass from its solution in chloroform were measured and then the surface free energy γ S of the layers was calculated from van Oss’ (LWAB) and Chibowski's (CAH) models. Also the images of the surfaces from SEM, confocal and optical microscopes, and profilometer were obtained. It was found that the surface of cholesterol pellets is more hydrophobic than this of the deposited layers, even if up to 200 statistical monolayers have been deposited on glass surface. From the images it was seen that the deposited layers were not uniform and at a larger coverage the needle-like cholesterol crystals were formed on the surface. From the zeta potential of cholesterol as a function of pH in 10 −3 M NaCl was found that cholesterol shows the isoelectric point at pH 2.65, which is within the range of the literature data 1.8–3. The hydroxyl ions OH – are the potential determining and probably adsorb on OH groups of cholesterol molecules. In the pH range 3–6 the literature and our results of the negative zeta potential of cholesterol are convergent, except for those measured on a cholesterol suspension precipitated in water from its solution in 1-propanol. The zeta potential of silica particles covered with statistical monolayer of cholesterol lie between those of pure silica and cholesterol suspensions. The calculated electrokinetic charge at the cholesterol surface is very low, e.g. at pH 11, where ζ = −50 mV, it amounts one excess negative charge (OH − ion) on the surface of 73 cholesterol molecules ( σ d = 0.422 μC/cm 2 ). The results obtained in this paper partially confirm those obtained previously and bring new insight into the structures of cholesterol layers deposited on glass.