The interactions of 1-dodecyl-azacycloheptan-2-one (Azone®), a penetration enhancer, with mouse skin were analyzed by fluorescence microscopy, solid-state ¹³C-CP/MAS-NMR spectroscopy and Attenuated Total Reflectance Fourier-transform infrared (ATR–FT-IR) spectroscopy. Ferulic acid was employed as fluorescent probe to observe the delivery pathway in the stratum corneum (SC) after treatment with Azone®. Results suggested that the interaction between Azone® and the SC occurs in the lipid domains as well as the protein domains. FT-IR measurements show that treatment with Azone® results in significant shifts toward larger wavenumbers at v _asCH₂ and v _sCH₂, indicating an increased gauche conformational isomer content of lipid CH₂. Further, a decrease of ^13CT₁ values and a shift of the SC protein amide-II band to a short wavenumber were found when the SC was pretreated with Azone®. It is concluded that Azone® can partially convert the SC protein from an α-helix conformation to a β-sheet conformation and loosen the aggregating SC keratins at room temperature.