Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H+-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H+-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H+ cotransport system is identical to the intestinal fluorescein/H+ cotransport system.