The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of NHE3 in LLC-PK1 cells. We found that NHE3-mediated Na+-dependent intracellular pH recovery decreased approximately 50% after 30 minute-treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: (1) The PKA antagonist H89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4 induced NHE3 inhibition. (2) The MEK1/2 inhibitor U0126 abolished the effect of the EPAC activator but only diminished the exendin-4 induced NHE3 inhibition. (3) Combination of H89 and U0126 fully prevented the effect of exendin-4 on NHE3. (4) No additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.