We evaluated the interaction between human stratum corneum (SC) and surfactant-based rigid or elastic vesicles during permeation using synchrotron X-ray diffraction to obtain the mechanism action of surfactant-based vesicles for enhanced skin permeation. The effects of vesicle elasticity on the interaction with SC were also investigated. Changes in the small-angle X-ray diffraction peaks of the human SC after buffer control and vesicle application were monitored. In the small-angle region, the control as the citrate buffer induced no significant changes of diffraction peaks for the lamellar structure of short periodicity phase (SPP), which is observed as a main peak in the human SC. Application of rigid vesicles resulted in small changes in the diffraction peaks attributed to lamellar phase of SPP. After application of elastic vesicles, however, a large shift to smaller angles due to swelling of the lamellar phase of SPP was clearly observed from the intensity difference profiles. All peaks due to the vesicles were still observed after 2 h of application for all formulations, indicating that both vesicles interacted with the SC while maintaining their structures. These results strongly suggest that vesicles affect the lamellar phase of SPP of the intercellular lipids in the SC during permeation.