While lithium is known to stimulate glucose transport into skeletal muscle, the mechanisms of the increased glucose transport by lithium in skeletal muscle are not well defined yet. We excised epitrochlearis muscles from male Wistar rats and measured the transport rates of a glucose analog into lithium-, insulin-, and muscular contraction-stimulated skeletal muscle tissue and we also analyzed the levels of cell surface glucose transport 4 using a photolabeling and multicolor immunofluorescence method. In addition, we generated a cell line that stably expresses myc-tagged GLUT4 to measure the rates of GLUT4 internalization and externalization. Lithium significantly increased 2-DG glucose transport rate in skeletal muscles; however, it was significantly lower than the stimulation induced by the maximum concentration of insulin or tetanic contraction. But co-treatment of lithium with insulin or tetanic contraction increased glucose transport rate by ∼200% more than lithium alone. When skeletal muscle tissues were treated with lithium, insulin, and muscular contraction, the levels of cell surface GLUT4 protein contents were increased similarly by ∼6-fold compared with the basal levels. When insulin or lithium stimuli were maintained, the rate of GLUT4myc internalization was significantly lower, and lithium was found to suppress the internalization of GLUT4myc more strongly. The lithium-induced increase in glucose uptake of skeletal muscles appears to increase in cell surface GLUT4 levels caused by decreased internalization of GLUT4. It is concluded that co-treatment of lithium with insulin and muscular contraction had a synergistic effect on glucose transport rate in skeletal muscle.