To evaluate the role of several specialized mechanisms for D-glucose transport in human oral mucosa, a cultured stratified cell layer derived from human oral mucosa was employed. Although this culture system has been used for reconstructive surgery, we, for the first time, tried to apply this system to the evaluation of nutrients and drug transport. Cell number and transepithelial electrical resistance (TEER) reached steady state 7-8 days after inoculation on the Transwell and TEER values at steady state were 130-140 ohm cm2, which was higher or lower than that of small intestine or Caco-2 cells, respectively. The transport studies were carried out using the cultured epithelium on the Transwell. The transport of D-glucose across the cultured stratified layer of oral epithelial cells was much more extensive than L-glucose, and was inhibited by 2-deoxy-D-glucose, a substrate of facilitative glucose transporters, and 2-methyl-D-glucoside, a specific substrate of a Na/glucose cotransporter (SGLT1). The results indicate that the sugar transporters function not only to take up D-glucose by the epithelial cells but also to transport the sugar across the stratified epithelial layer.