Duodenocyte pHi control and HCO3 − secretion protects the proximal duodenum against damage by gastric acid. The molecular details of duodenocyte pH control are not well understood. A selective duodenal expression (within the upper GI tract) has been reported for the electroneutral Na+:HCO3 − cotransporter NBCn1 (Slc4a7). We aimed to determine the role of NBCn1 and NBCe2 in duodenocyte intracellular pH regulation as well as basal and agonist-stimulated duodenal bicarbonate secretion (JHCO3 −), exploiting mouse models of genetic slc4a7 and slc4a5 disruption. Basal and forskolin (FSK)-stimulated JHCO3 − was measured by single-pass perfusion in the duodenum of slc4a7−/− and slc4a7+/+ as well as slc4a5−/− and slc4a5+/+ mice in vivo, and by pH-stat titration in isolated duodenal mucosa in vitro. Duodenocyte HCO3 − uptake rates were fluorometrically assessed after acidification of intact villi and of isolated duodenocytes. Slc4a7−/− mice displayed significantly lower basal and FSK-stimulated duodenal HCO3 − secretion than slc4a7+/+ littermates in vivo. FSK-stimulated HCO3 − secretion was significantly reduced in slc4a7−/− isolated duodenal mucosa. Na+- and HCO3 −-dependent base uptake rates were significantly decreased in slc4a7−/− compared with slc4a7+/+ villus duodenocytes when measured in intact villi. Carbonic anhydrase (CA)-mediated CO2 hydration played no apparent role as a HCO3 − supply mechanism for basal or FSK-stimulated secretion in the slc4a7+/+ duodenum, but was an important alternative HCO3 − supply mechanism in the slc4a7−/− duodenum. NBCe2 (Slc4a5) displayed markedly lower duodenal mRNA expression levels, and its disruption did not interfere with duodenal HCO3 − secretion. The electroneutral Na+:HCO3 − cotransporter NBCn1 (slc4a7) is a major duodenal HCO3 − importer that supplies HCO3 − during basal and FSK-stimulated HCO3 − secretion.