Lipid lamellar structure in intercellular space of stratum corneum is recognized as main physical barriers against dehydration and invasion of foreign substances. For development of effective transdermal drug delivery, it is necessary to decrease those barriers temporally and reversibly. Co-administration of components which has drug absorption enhancing effect is one of the essential methodologies to increase absorption of drugs via skin. The change in lipid lamellar structure was determined by synchrotron X-ray diffraction. When the stratum corneum was pretreated by l-menthol, lipid lateral packing became liquid crystal. It was considered that disorder of lamellar structure brought about increasing in drug permeation. It means that the lipid lamellar structure is one of the key parameters to evaluate physical barrier function of the skin. The structure analysis of lipid lamellar employing synchrotron X-ray diffraction is thought to be a meaningful technique possibly applied for promoting mechanism of transdermal absorption enhancer and evaluation of barrier function of skin.