We investigated the role of the Na⁺/H⁺ exchanger 3 (NHE3) regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane morphology and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as a attenuated Na⁺ absorption in isolated mucosa of jejunal and colonic tissue but not of ileal mucosa in vitro. However, cAMP- mediated inhibition of both parameters remained intact. The basal NHE3 transport rate was decreased in surface colonocytes, while inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the brush border membrane (BBM) of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated membranes from the small and large intestine. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adapter proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.