There are several data concerning transporters expression and/or regulation in cell lines maintained in different conditions, such as medium glucose concentration. This work aimed to evaluate the influence of two different extracellular glucose concentrations, commonly used in culture media, on the intestinal absorption of organic cations. Thus, the effect of 5.5 mM glucose and 25 mM glucose (HG) in culture media, was studied on [(3)H]-MPP(+) (1-methyl-4-phenylpyridinium iodide) uptake in Caco-2 cells. Expression of human organic cation transporter type 1 (hOCT1) and human organic cation transporter type 3 (hOCT3) was investigated in cells cultured at both glucose concentrations. [(3)H]-MPP(+) uptake, as well as its affinity for the transporter, were significantly decreased in HG cells. Moreover, hOCT3 mRNA levels were reduced in HG cells. Functional confirmation of this result was made using hOCT3 inhibitors. In conclusion, maintenance of Caco-2 cells (commonly used in several in vitro studies on membrane transport) in HG conditions affects organic cation transport at the intestinal level. Hence, results obtained in these conditions must be analysed with great care, since extracellular glucose levels may originate changes in organic cation nutrient and drug bioavailability.