Prolonged fructose consumption in rodents stimulates intestinal expression of transporters and enhances intestinal sugar absorption. These dietary conditions also induce peripheral insulin resistance. Intestinal sugar transport into enterocytes is controlled by transient expression of GLUT2 in the brush border membrane (BBM) after a sugar load. Sugars target GLUT2 to the BBM (reviewed in Kellett & Brot-Laroche, Diabetes, 2005; 54;3056-3062). Sugar ingestion increases plasma insulin and might regulate BBM GLUT2 location. The aim of this study was to investigate the potential role of insulin on GLUT2 trafficking in entocytes in health and disease. In glucose-fed Caco2/TC7 enterocytes, GLUT2 was found in the BBM. Insulin addition to culture media inhibited BBM GLUT2 location as shown by surface biotynylation and immunofluorescence labelling. Therefore insulin signalling is perceived by enterocytes in the absence of other changes in cell environment. Insulin decreased BBM GLUT2 location and fructose transport in the jejunum of hyperinsulinemic-euglycaemic (HIEG) clamped mice. Decreased BBM GLUT2 lowered sugar uptake. Moreover, mice fed fructose for more than 3 weeks exhibited systemic insulin resistance and high BBM GLUT2 that could not be removed in HIEG clamps. Thus insulin resistance produces insulin insensitive BBM GLUT2 trafficking. Alike diabetic patients who perform insulin injections prior to meals, insulin injection to mice prior an oral fructose load prevented BBM GLUT2 translocation into the BBM. These results obtained in vivo and in vitro indicate that GLUT2-mediated sugar absorption in the intestine is controlled by insulin. We thus propose that insulin exerts, part of its hypoglycaemic effect by slowing down dietary sugar absorption, a mechanism that would limit glucose excursions in the blood stream.