Liquid crystal (LC) emulsions composed of multi-lamellar structures were formulated to enhance the percutaneous absorption of retinyl palmitate across skin barriers. The LC emulsions were prepared by hot process emulsification of an aqueous phase and an oil phase containing retinyl palmitate. Percutaneous absorption levels across synthetic membranes were determined by Franz cell diffusion study. The diffusional absorption level of the oil phase in the LC emulsions was also visualized by fluorescence imaging of Nile red-containing emulsions on synthetic membranes. Transport of retinyl palmitate across pig skin was fluorescently visualized. In vitro cell culture study showed that the LC emulsions enhanced cellular metabolism and proliferation of fibroblasts in comparison to the plain emulsion. The multi-lamellar structures composed of aligned fatty alcohol chains were revealed using freezefracture scanning electron microscopy and polarized light microscopy. The LC emulsions showed increased retention at the membrane as well as at the acceptor in comparison to plain oil-in-water emulsions. Total fluorescence intensity at the membrane with the LC emulsions was higher compared to plain emulsions. Confocal laser scanning microscopy (CLSM) of cryo-cut cross-sections revealed that Nile red was highly localized in the epidermis layers, particularly in the stratum corneum in comparison to the plain emulsions. LC emulsions also enhanced transport of retinyl palmitate at cellular levels, in addition to facilitating percutaneous absorption across dermal layers.