Background: The human facilitative glucose transporter GLUT12 was isolated from the breast cancer cell line MCF-7 (Rogers, S et al. 2002). GLUT12 is expressed in human crude membranes of adipose tissue, small intestine and skeletal muscle, where it seems to act as a secondary insulin-sensitive transporter. We have demonstrated in Xenopus laevis oocytes expression system that hGLUT12 can transport α-methyl-glucoside (αMG), a specific SGLT substrate, and that this transport is enhanced in the presence of Na+ (Pujol-Gimenez et al. 2015). Based on this information, the aim of the present work was to investigate the location and function of GLUT12 in small intestine. Methods: GLUT12 location in human and rat small intestine was investigated by immunohistochemical methods. The samples were part of the department collection that had been collected for previous studies and approved by the Ethics Committee of the University of Navarra. Samples were fixed in bouin and the antibody anti-GLUT12 used at 1:100. Expression of GLUT12 in brush border membrane vesicles (BBMV) of Caco-2 cells and its regulation by different sugars was studied by Western blot using the same antibody at 1:1000. Uptake experiments were performed in Caco-2 cells grown on 24 well plates. Cells were incubated for 15 min with 5 mM αMG (with traces of the radiolabel sugar) in the presence and absence of Na+ and in the presence of the GLUT12 substrates fructose or 2-deoxy-glucose (2-DOG) at 50 mM. Regulation of GLUT12 activity by PKC and PKA was investigated measuring αMG uptake (5 mM, 15 min) after 60 min pre-incubation of the cells with PMA (0.1 µM) or Forskolin (10 mM). Regulation of GLUT12 activity by the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) was investigated by Western blot and uptake assays after 1hour of pre-incubation of the cells with 10 or 25 ng/ml TNF-α. Results: Immunolabeling for GLUT12 appeared in the apical cytoplasm, below the brush border of rat and human enterocytes; in the perinuclear region of human enterocytes and in rat basolateral cytoplasm. Western blot analysis showed GLUT12 expression in the BBMV of Caco-2 cells. This expression was upregulated by incubation of the cells with glucose, galactose, fructose and αMG. In the absence of Na+, αMG uptake was inhibited by 2-DOG, a specific substrate of the GLUTs transporters. Transport of αMG was up-regulated by PKC but not by PKA. TNF-α increased αMG uptake by Caco-2 cells. This increase was accompanied by translocation of GLUT12 to the apical membrane. Conclusion: These results demonstrate the expression, functional activity and regulation of GLUT12 in the apical membrane of enterocytes, opening new venues to investigate its role in physiological and pathophysiological conditions in the intestine.