We hypothesized that GLUT12 is involved in regulation of glucose flux in distal renal tubules in response to elevated glucose. We used the MDCK polarized epithelial cell model and neutralizing antibodies to analyze GLUT12 targeting and directional GLUT12 mediated glucose transport. At physiological glucose concentrations, GLUT12 was localized to a perinuclear position. High glucose and serum treatment resulted in GLUT12 localization to the apical membrane. This mitogen stimulated targeting of GLUT12 was inhibited by rapamycin, the specific inhibitor of mTOR. The functional role of GLUT12 was also examined. We constructed a GLUT12 cDNA containing a c-Myc epitope tag in the fifth exofacial loop. Assays of glucose transport at the apical membrane were performed using Transwell filters. By comparing transport assays in the presence of neutralizing anti-c-Myc monoclonal antibody we specifically measured GLUT12 mediated glucose transport at the apical surface. GLUT12 mediated glucose transport was mitogen dependent and rapamycin sensitive. Our results implicate mTOR signaling in a novel pathway of glucose transporter protein targeting and glucose transport. Activity of the mTOR pathway has been associated with diabetic kidney disease. Our results provide evidence for a link between GLUT12 protein trafficking, glucose transport and signaling molecules central to the control of metabolic disease processes.