Backround: Inhibition of the apical Na⁺/H⁺ exchanger isoform 3 (NHE3) activity interferes with dipeptide uptake in intestinal cell lines. Aim: This study investigates how dipeptide transport affects murine jejunal fluid absorption and enterocyte pHi in vivo, whether NHE3 function is mandatory for PepT1 function in the native intestine, and whether the reason for its importance lies in its ability to regulate enterocyte pHi during H+/dipeptide uptake. Methods and Results: The luminal application of Gly-Sar resulted in a strong, PepT1- as well as NHE3-dependent increase in intestinal fluid secretion, measured by single-pass perfusion, and a decrease in villous enterocyte pHi, measured by two-photon microscopy in the anesthetized mouse (administration of 10 µL/g intraperitoneal (IP) haloperidol/midazolam/fentanyl cocktail (haloperidol 12.5 mg/Kg, fentanyl 0.325 mg/Kg and midazolam 5 mg/Kg body weight)). It also caused a strong short circuit current (Isc) response in chambered jejunal mucosa in vitro, which was not influenced by the absence or presence of the luminal Cl^–/HCO₃^– exchanger SLC26a6, was absent in the absence of PepT1 expression, and was virtually abolished in the absence of NHE3 expression. Villous enterocyte pHi decreased significantly stronger upon the luminal application of Gly-Sar in the absence of Slc26a6, as well as in the absence of NHE3. The absence or inhibition of NHE1 and NHE2, which are also expressed in murine jejunum, did not affect the Gly-Sar mediated pHi-decrease. Conclusions: NHE3 and Slc26a6 are equally involved in pHi regulation after PepT1-mediated uptake of H+/dipeptide over the apical BBM, but only NHE3 is essential for both PepT1-mediated dipeptide as well as salt and fluid absorption. The requirement of NHE3 for PepT1-mediated transport may be explained by a unique H⁺-recycling function of NHE3 in conjunction with PepT1.