Structural properties of the cholesteryl acetate have been investigated by Raman spectroscopy. In this work, we present Raman spectroscopy studies of the C=O stretching mode for this eater of cholesterol in the crystalline and isotropic liquid phases. Our interpretation of the structural organization is compared with some semi-empirical quantum calculations. A hypothesis is suggested in order to explain the difference between our Raman spectroscopy investigations and the X-ray results.