Intestinal epithelial cells undergo differentiation as they move from the crypt to the villi, a process that is associated with up- and downregulation in expression of a variety of genes, including those involved in nutrient absorption. Whether the intestinal uptake process of vitamin B(2) [riboflavin (RF)] also undergoes differentiation-dependent regulation and the mechanism through which this occurs are not known. We used human-derived intestinal epithelial Caco-2 cells and native rat intestine as models to address these issues. Caco-2 cells showed a significantly higher carrier-mediated RF uptake in post- than preconfluent cells. This upregulation was associated with a significantly higher level of protein and mRNA expression of the RF transporters hRFVT-1 and hRFVT-3 in the post- than preconfluent cells; it was also accompanied with a significantly higher rate of transcription of the respective genes (SLC52A1 and SLC52A3), as indicated by the higher level of expression of heterogeneous nuclear RNA and higher promoter activity in post- than preconfluent cells. Studies with native rat intestine also showed a significantly higher RF uptake by epithelial cells of the villus tip than epithelial cells of the crypt; this again was accompanied by a significantly higher level of expression of the rat RFVT-1 and RFVT-3 at the protein, mRNA, and heterogeneous nuclear RNA levels. These findings show, for the first time, that the intestinal RF uptake process undergoes differentiation-dependent upregulation and suggest that this is mediated (at least in part) via transcriptional mechanisms.