Plasma glutamate concentrations are constant despite dynamic changes in diets. Most likely, virtually all the dietary glutamate is metabolized in the gut. The present study investigated permeability and metabolism of dietary glutamate in a Caco-2 intestinal epithelial cell layer model by tracing the fate of [U-13C] or [15N]glutamate added to the apical medium. For comparison, several other labelled essential and non-essential amino acids were tested as well. Almost all the labelled glutamate in the apical medium (98% and 96% at 24 h of the culture, respectively) was incorporated in the cell layer, while it barely appeared at the basolateral side, indicating an almost complete utilization of glutamate. Indeed, the 13C was incorporated into alanine, proline, ornithine, and glutamine, and the 15N was incorporated into alanine, glutamine, ornithine, proline, branched chain amino acids and also found as ammonia indicative of oxidation. In contrast, substantial apical-to-basolateral transport of amino acids (8–85% of uptake) other than glutamate and aspartate was evident in studies using amino acid tracers labelled with 13C, 15N or D. These results suggest that the intestinal epithelial cell monolayer utilizes dietary glutamate which adds to maintaining glutamate homeostasis in the body.