Skeletal muscle is the main tissue responsible for glucose utilization in the fed state, and it expresses the ubiquitous GLUT1 glucose transporter and the muscle/fat specific GLUT4 glucose transporter. Here we investigated the expression of these transporters during muscle cell differentiation in vitro. Rat L6 muscle cells were grown to the stages of myoblasts, alignment and fused myotubes. Glucose (2-deoxy-D-glucose) transport was higher in myoblasts, decreasing with the progression of alignment and cell fusion. Conversely, insulin-stimulated glucose uptake was negligible in myoblasts, and increased with cell alignment and fusion. The cellular content of GLUT1 transporters decreased and that of GLUT4 transporters increased with cell fusion. Insulin rapidly stimulated glucose uptake in fused myotubes maintained in 2% serum but not in 10% serum. In 10% serum, basal glucose uptake increased as did the cellular content of GLUT1 transporters, while GLUT4 transporter content did not change. These results indicate that both transporters are regulated oppositely during muscle cell differentiation, and that high serum concentrations override the capacity of insulin to regulate transport by inducing overexpression of the GLUT1 transporter.