The stratum corneum (SC) consists of a lipid layer that forms two types of lamellar structures: short lamellar (S-La) and long lamellar (L-La). It has been reported that S-La contains water phases in the hydrophilic region of the lipids, and that it may play an important role in regulating the water content of the SC. The amount of water in the SC can affect how a drug carrier permeates through the intercellular lipid pathway. To better understand the impact of SC water content on the skin penetration mechanism of a microemulsion (ME), we conducted a study using small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), and small-angle neutron scattering (SANS). Our results showed that MEs can enhance skin permeation under humid conditions because the lipid packing structures of the hydrated SC are more disrupted than those of the dry SC. The results also showed that the inner water of MEs was released to the SC when applying MEs to the dry SC, resulting in an increase in the repeat distance of S-La. Conversely, when MEs are applied to hydrated SC, the MEs absorb the water from the SC into their inner phases, causing a decrease in the repeat distance of S-La over time.