Various in vivo studies have investigated the insulin response that is elicited when glutamate is elevated in circulation or in a given tissue; fewer studies have investigated the effects of glutamate on glucose uptake and handling. Glutamate ingestion in humans can attenuate rises in blood glucose following a carbohydrate load in the absence of increases in serum insulin concentrations. However, the underlying mechanisms have yet to be investigated. To elucidate the effects of glutamate on glucose handling in skeletal muscle tissue, differentiated rat L6 myocytes were treated with glutamate, and glucose uptake was assessed with the use of 2-[3H]-deoxy-d-glucose ([3H]-2-DG). Cells treated with 2 mmol/L glutamate experienced the greatest increase in [3H]-2-DG uptake relative to the control condition (177% ± 2% of control, P < 0.001) and the uptake was similar to that of metformin (184% ± 4%, P < 0.001). In line with these findings, differentiated glucose transporter 4 (GLUT4)-overexpressing myotubes treated with 2 mmol/L glutamate displayed significantly increased GLUT4 translocation when compared with the control condition (159% ± 8% of control, P < 0.001) and to an extent similar to that of insulin and metformin (181% ± 7% and 159% ± 12%, respectively). An AMP-activated protein kinase (AMPK) inhibitor (Compound C) abolished the glutamate-stimulated glucose uptake (98% ± 12% of control), and Western blotting revealed significantly elevated AMPK phosphorylation (278% ± 17% of control, P < 0.001) by glutamate. Our findings suggest that when muscle cells are exposed to increased glutamate concentrations, glucose uptake into these cells is augmented through AMPK activation, through mechanisms distinct from those of insulin and leucine.