Up to now, the molecular mechanism of the penetration enhancing effect of sucrose esters (SEs) on stratumcorneum (SC) has not been explained in details. In this study, variety of surface sensitive techniques, including surface pressure-area (π-A) isotherms, infrared reflection-absorption spectroscopy (IRRAS), and Brewster angle microscopy (BAM), have been used to investigate interactions between SEs and SC intercellular lipids. A monolayer of the mixture of ceramide AS C18:18, stearic acid and cholesterol in the molar ratio of 1:1:0.7 on an aqueous subphase is a good model to mimic a single layer of intercellular SC lipids. The π-A isotherms of mixed monolayers and parameters derived from the curves demonstrated the interaction between nonionic surfactants such as SEs and SC lipids. With increasing SE concentration, the resultant monolayer films became more fluid and better compressible. IRRAS measurements showed that SEs disordered the acyl chains of SC lipids, and the BAM images demonstrated the modification of the domain structures in SC monolayers. Longer chain-SE has a stronger disordering effect and is better miscible with ceramides in comparison to SE with a shorter hydrophobic part. In conclusion, this study demonstrates the disordering effect of SEs on the biomimetic SC model, pointing out that small changes in the structure of surfactant may have a strong influence on a penetration enhancement of lipophilic drugs through intercellular lipids of skin.