Desmosterol is a direct biosynthetic precursor of cholesterol in Bloch biochemical pathway of cholesterol biosynthesis and differs with cholesterol only by a double bond in carbon 24. In this study, we aimed to research for the first time comparative effects of cholesterol and its precursor desmosterol on dipalmitoyl phosphatidylcholine (DPPC) multilamellar vesicles (MLVs) by utilizing Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Our DSC studies reveal that with the addition of increasing desmosterol and cholesterol concentrations into pure DPPC MLVs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears. While the main phase transition is abolished at 25 mol% concentration of desmosterol, this disappearance of the main phase transition occurs at cholesterol concentration above 30 mol%. Our FTIR studies show that both desmosterol and cholesterol decrease the order in the gel phase, whereas they increase it in the liquid crystalline phase. Importantly, we found that the effect of desmosterol on membrane order is weaker than that of cholesterol in both phases. Moreover, desmosterol and cholesterol increase the dynamics of DPPC membranes in the gel phase, while they decrease it in the liquid crystalline phase. Both sterols also induce a decrease in the wavenumber values of the C=O stretching and PO2(-) antisymmetric double stretching bands of DPPC both in the gel and liquid crystalline phases, which points out hydrogen bonding in between the hydroxyl group of both sterols and the carbonyl and phosphate groups of DPPC membranes.