Apical membrane sodium hydrogen exchanger-3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na⁺ absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein, synaptotagmin I (Syt I), plays a pivotal role in cAMP- and Ca⁺⁺-induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco2_bbe Syt I co-immunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca⁺⁺. Following siRNA suppression of Syt I expression, cAMP and Ca⁺⁺-induced inhibition of NHE3 activity were still observed, but NHE3 endocytosis was blocked, assessed by ²²Na influx and the apical membrane biotin-labeling respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the mu subunit of the adaptor protein 2 complex or the heavy chain of clathrin. Co-immunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca⁺⁺-induced NHE3-Syt I interaction preceded and was required for recruitment of AP-2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca⁺⁺-induced endocytosis of NHE3 (but not in inhibition of activity), through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.