Abstract To develop a transdermal drug delivery system, it is necessary to search for effective modulators to act as permeation enhancers and evaluate its mechanisms of action. It has been suggested that attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) is a useful tool for evaluating the functional group interaction of the intercellular lipids in the stratum corneum. The purpose of this study is elucidation of the effect of the transdermal permeation enhancers on the intercellular lipid in hairless rat stratum corneum using the ATR-FTIR. Firstly, to confirm the frequencies related to the intercellular lipid in stratum corneum, CH 2 asymmetric and symmetric vibrations were clearly related to the intercellular lipids. In intact stratum corneum, the blue shift of CH 2 asymmetric and symmetric stretching vibrations begins at about 40 °C and remarkable change is induced at 50 °C. The administration of l-menthol causes disorder of the intercellular lipids in stratum corneum similar to that of heat application. The disordering of intercellular lipid lattices in stratum corneum induced by the l-menthol might be related to the enhancing effect of l-menthol. The results provide information for the development of novel transdermal drug delivery systems.