Oligopeptide transporter PEPT1 is thought to be involved in the intestinal absorption and renal reabsorption of peptides and therapeutic agents. The driving force of PEPT1 is H+ gradient, a part of which is supplied by Na+/H+ exchanger (NHE) expressed on the apical surface of the epithelium although molecular identification of NHE has not yet been fully clarified. Here we examined the effect of NHE3 coexpression on the function of PEPT1 to support the hypothesis that NHE3 regulates PEPT1 function by supplying its driving force. HEK293 cells expressing PEPT1 alone exhibited Na+-independent but pH-dependent uptake of glycylsarcosine (GlySar), whereas those coexpress PEPT1 and NHE3 showed an increase in GlySar uptake and conferred Na+-dependence on the uptake of GlySar. The increase in GlySar transport by PEPT1 depended on the expression level of NHE3 and was found at various levels of PEPT1 expression. Kinetic analysis of GlySar uptake in HEK293 cells expressing both PEPT1 and NHE3 or those expressing PEPT1 alone revealed an approximately 3 times increase in the transport capacity in the presence of NHE3, as normalized by PEPT1 mRNA expression. Confocal microscopy indicated that both PEPT1 and NHE3 are colocalized on the cell-surface of HEK293 cells. Thus, the present findings are the first to specify that NHE3 exerts post-transcriptional stimulation of PEPT1-mediated transport and can affect cellular uptake of the substrates by PEPT1 expressed on apical membranes in the body.