This study investigates, for the first time, the ability of punicalagin to modulate intestinal glutamate uptake by upregulation of the expression of one of its transporters present on the enterocyte membrane. The use of an Ussing chamber revealed an increase in glutamate transport in differentiated Caco-2 cells after punicalagin treatment for 24 h. This cell line constitutively expresses two glutamate transporters: EAAT1 and EAAT3. In response to punicalagin, the expression of EAAT3 was increased, at both mRNA and protein levels, but not that of EAAT1. Transfection with EAAT3-targeting siRNA specifically altered basal and induced EAAT3 gene expression, decreasing the positive effect of punicalagin on glutamate uptake. These data confirmed the involvement of EAAT3 in increasing glutamate uptake by enterocytes after punicalagin treatment.