The major components of the intercellular lipids in the stratum corneum are ceramides (CERs), cholesterol (CHOL) and free fatty acids. To estimate the effects of l-menthol on the intercellular lipids, we elucidated the thermotropic behavior and lateral structure of a CER2/CHOL mixture. This formed a eutectic mixture at molar fraction of CHOL (Xch) = 0.6, as a lamellar structure with a repeat distance of 3.99 nm, and it showed a hexagonal hydrocarbon chain packing conformation at ambient temperature. In thermograms of the CER2/CHOL mixtures, the addition of l-menthol broadened the transition peaks and reduced the enthalpy of transition. The effect of l-menthol on the change in transition temperature of the eutectic mixture at Xch = 0.6 was weaker than for other molar fractions of CHOL. This suggested that the structure of the eutectic mixture was stable to the effects of l-menthol. Furthermore, l-menthol increased the repeat distance of the lamellar structure and inhibited the formation of hexagonal hydrocarbon chain packing. Therefore, l-menthol induced a disturbance of the lateral structure of the eutectic mixture of CER2/CHOL and the phase transition became unclear. These effects of l-menthol could enhance transdermal drug delivery.