PAT1 (Slc26a6) is located on the apical membrane of the small intestinal villi, but its role for salt absorption has not been studied. To ascertain the role of Slc26a6 in jejunal sodium and chloride absorption, and its interplay with NHE3, muscle-stripped jejuna from Slc26a6+/+ and −/− and NHE3 +/+ and −/− mice were mounted in Ussing chambers and electrical parameters, and ³⁶Cl⁻ and ²²Na⁺ fluxes were measured. In parallel studies, expression of the apical Na⁺/H⁺ exchanger (NHE3) was examined by immunofluorescence labeling and immunoblot analysis in brush border membrane (BBM). In the basal state, net Cl⁻ and Na⁺ fluxes were absorptive in Slc26a6−/− and +/+ jejuni, but significantly decreased in −/− animals. Upon forskolin addition, net Na⁺ absorption decreased, Isc strongly increased, and net Cl⁻ flux became secretory in Slc26a6−/− and +/+ jejuni. When luminal glucose was added to activate Na⁺/glucose cotransport, concomitant Cl⁻ absorption was significantly reduced in Slc26a6 −/− jejuni, while Na⁺ absorption increased to the same degree in Slc26a6 −/− and +/+ jejuni. Identical experiments in NHE3-deficient jejuni also showed reduced Na⁺ and Cl⁻ absorption. Results further demonstrated that the lack of NHE3 rendered Na⁺ and Cl⁻ absorption unresponsive to inhibition by cAMP, but did not affect glucose-driven Na⁺ and Cl⁻ absorption. Immunoblotting revealed comparable NHE3 abundance and distribution in apical membranes in Slc26a6−/− and +/+ mice. The data strongly suggests that Slc26a6 acts in concert with NHE3 in electroneutral salt absorption in the small intestine. Slc26a6 also serves to absorb Cl⁻ during glucose-driven salt absorption.