Na+/H+ exchanger 3 (NHE3), a major Na+ transporter in the luminal membrane of proximal tubule, is subject to angiotensin II (ANG II) regulation in renal Na+/fluid absorption and blood pressure control. We have previously shown that inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) mediates ANG II-induced exocytosis of NHE3 in cultured proximal tubule epithelial cells. In searching for scaffold protein(s) that coordinates with IRBIT in NHE3 trafficking, we found that Na+/H+ exchanger regulatory factor 1 (NHERF1), NHE3 and IRBIT proteins were co-expressed in the same macrocomplexes and loss of type 1 receptor of angiotensin II (AT1R) decreased their expression in the renal brush border membrane. We found that NHERF1 was required for ANG II-mediated forward trafficking and activation of NHE3 in cultured cells. ANG II induced a concomitant increase of NHERF1 interaction with NHE3 and IRBIT, which was abolished when NHERF1 PDZ1 domain was removed. Overexpression of an YFP-NHERF1 construct that lacks PDZ1, but not PDZ2, failed to exaggerate ANG II-dependent increase of NHE3 expression in the apical membrane. Moreover, exogenous expression of PDZ1 exerted a dominant-negative effect on NHE3 activation by ANG II. We further demonstrated that IRBIT was indispensable for ANG II-provoked increase in NHERF1-NHE3 interaction, and that phosphorylation of IRBIT at Ser68 was necessary for the assembly of the NHEF1-IRBIT-NHE3 complex. Taken together, our findings suggest that NHERF1 mediates ANG II-induced activation of renal NHE3, which requires coordination between IRBIT and NHERF1 PDZ1 domain in binding and transporting NHE3.