The interfacial tension between hexane solution of cholesterol and water was measured as a function of temperature and the mole fraction of cholesterol in hexane under atmospheric pressure. The interfacial tension vs. mole fraction of cholesterol curve was observed to have two break points at a certain temperature; the concentration of the break point increases with increasing temperature. The experimental results were analyzed by applying the rigorous thermodynamics of adsorption at interfaces developed previously. The excess number of moles of cholesterol and the thermodynamic quantity changes associated with its adsorption were found to show the discontinuous changes at the break points. It was concluded that the first-order phase transition takes place from the gaseous to the expanded state and from the expanded to the condensed one in the adsorbed film of cholesterol at the hexane/water interface.