Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca²⁺, which regulates ion transport, including bicarbonate secretion. However, the underlying Ca²⁺ handling mechanisms are poorly understood. The aim of the present study was to determine whether Na⁺/Ca²⁺+ exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and bicarbonate secretion by controlling Ca²⁺ homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current (I_sc) and bicarbonate secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot and cytosolic Ca²⁺ in duodenocytes was measured by fura-2. Carbachol (100 µM) increased I_sc in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced bicarbonate secretion peaked at 10 min. 2-aminoethoxydiphenylborate (2-APB, 100 µM) or LiCl (30 mM) significantly reduced the initial peak in I_sc by 51% or 47%, respectively, and abolished the plateau phase of I_sc without affecting bicarbonate secretion induced by carbachol. Ryanodine (100 µM), caffeine (10 mM) and nifedipine (10 µM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10-30 µM) significantly inhibited carbachol-induced increases in duodenal mucosal I_sc and bicarbonate secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae and functional NCX in duodenocytes was demonstrated in Ca²⁺ imaging experiments where Na⁺ depletion elicited Ca²⁺ entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca²⁺-dependent duodenal mucosal ion transport and bicarbonate secretion that results from stimulation of muscarinic receptors.