Abstract  The duodenal villus brush border membrane expresses several ion transporters and/or channels, including the solute carrier 26 anion transporters Slc26a3 (DRA) and Slc26a6 (PAT‐1), the Na+/H+ exchanger isoform 3 (NHE3), as well as the anion channels CFTR and Slc26a9. Using genetically engineered mouse models lacking Scl26a3, Slc26a6, Slc26a9, or Slc9a3 (Na+/H+ exchanger isoform 3, NHE3), the study was carried out to assess the role of these transporters in mediating the protective duodenal HCO3− secretory response (DBS‐R) to luminal acid; and to compare it to their role in DBS‐R elicited by the adenylyl cyclase agonist forskolin (FSK). While basal DBS was reduced in the absence of any of the three Slc26 isoforms, the DBS‐R to FSK was not altered. In contrast, the DBS‐R to a 5 min exposure to luminal acid, pH 2.5, was strongly reduced in the absence of Slc26a3 or Slc26a9, but not Slc26a6. CFTR inhibitor [CFTR(Inh)‐172] reduced the first phase of the acid‐induced DBS‐R, while NHE3 inhibition (or knockout) abolished the sustained phase of the DBS‐R. Luminal acid exposure resulted in the activation of multiple intracellular signalling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by endocytosis in the later stage. We conclude that the long‐lasting DBS‐R to luminal acid exposure activates multiple duodenocyte signalling pathways and involves changes in trafficking and/or activity of CFTR, Slc26 isoforms Slc26a3 and Slc26a9, and NHE3. This article is protected by copyright. All rights reserved