Na+/H+ exechanger 3 (NHE3) plays an important role in neutral Na+ transport in mammalian epithelial cells. The Rho family of small GTPases and the PSD-95/discs large/ZO-1 (PDZ) domain-based adaptor Shank2 are known to regulate membrane expression and activity of NHE3. In this study the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, was examined in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2 and βPix form a macromolecular complex when expressed heterologously in PS120 fibroblastic cells as well as endogenously in rat colon, kidney and pancreas. The expression of these proteins was mostly confined to the apical region of rat colonic epithelial cells in immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. The effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor. These indicate that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix upregulates membrane expression and activity of NHE3 by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.