Riboflavin transporter (RFT) 2 has recently been identified as a transporter that may be, mainly based on the functional characteristics of its rat ortholog (rRFT2), involved in the intestinal absorption of riboflavin. The present study was conducted to further examine such a possible role of RFT2, focusing on the functional characteristics of its human ortholog (hRFT2) and the response of rRFT2 expression in the small intestine to deprivation of dietary riboflavin. When transiently expressed in human embryonic kidney 293 cells, hRFT2 could transport riboflavin efficiently in a pH-sensitive manner, favoring acidic pH and without requiring Na(+). Riboflavin transport by hRFT2 was saturable with a Michaelis constant of 0.77 μmol/L at pH 6.0, and inhibited by some riboflavin derivatives, such as lumiflavin. It was also inhibited, to a lesser extent, by some cationic compounds, such as ethidium. Thus, hRFT2 was suggested to, together with a finding that its mRNA is highly expressed in the small intestine, have characteristics as an intestinal RFT. Furthermore, feeding rats a riboflavin-deficient diet caused an upregulation of the expression of rRFT2 mRNA in the small intestine, presumably as an adaptive response to enhance riboflavin absorption, which would involve rRFT2, and its apically localized characteristic was suggested by the observation of rRFT2 tagged with green fluorescent protein stably expressed in polarized Madin-Darby canine kidney II cells. All these results combined indicate that RFT2 is a transporter involved in the epithelial uptake of riboflavin in the small intestine for its nutritional utilization.