We have recently shown the co-localization of Rab11 and the glucose transporter GLUT4 in cardiac muscle and an insulin-stimulated increase of Rab11 in GLUT4-containing vesicles in this tissue. We now assessed the effect of Rab11 wt and a dominant-negative mutant (N124I) on GLUT4 trafficking in the cardiomyoblast cell line H9c2 stably overexpressing the insulin receptor (H9c2-E2) and in human primary skeletal myotubes. These cells were used for transient cotransfection or adenoviral co-infection with GLUT4myc and Rab11 wt or N124I with subsequent determination of 2-deoxyglucose (2-DOG) uptake and GLUT4myc translocation. Concomitant overexpression of GLUT4myc and Rab11 wt in cardiomyocytes decreased the amount of GLUT4myc at the cell surface by about 50%, an effect not observed for Rab11 N124I. However, the dominant-negative mutant reduced the efficiency of insulin to promote glucose uptake and GLUT4 translocation in both cardiac and skeletal muscle cells to about one half. The level of Akt phosphorylation does not vary after cotransfection indicating that insulin signalling remained unaffected under these conditions. In conclusion, our data show that Rab11 (i) mediates endocytosis of GLUT4 and (ii) plays a pivotal role in insulin-regulated translocation of this transporter to the plasma membrane.