Insulin- and contraction-mediated glucose transporter 4 (GLUT4) trafficking have different kinetics in mature skeletal muscle. Intravital imaging indicates that insulin-stimulated GLUT4 trafficking differs between t-tubules and sarcolemma. In contrast, contraction-induced GLUT4 trafficking does not differ between membrane surfaces. This distinction likely is caused by differences in the underlying signaling pathways regulating GLUT4 vesicle depletion, GLUT4 membrane fusion, and GLUT4 reinternalization.