Sodium-proton exchanger 3 (NHE3/SLC9A3) located in the apical membrane of renal and gastrointestinal epithelia mediates salt and fluid absorption and regulates pH homeostasis. As an auxiliary regulatory factor of NHE proteins, calcineurin B homologous protein 1 (CHP1) facilitates NHE3 maturation, plasmalemmal expression, and pH sensitivity. Dysfunctions of NHE3 are associated with renal and digestive system disorders. Here, we report the cryo–electron microscopy structure of the human NHE3-CHP1 complex in its inward-facing conformation. We found that a cytosolic helix-loop-helix motif in NHE3 blocks the intracellular cavity formed between the core and dimerization domains, functioning as an autoinhibitory element and hindering substrate transport. Furthermore, two phosphatidylinositol molecules are found to bind to the peripheric juxtamembrane sides of the complex, function as anchors to stabilize the complex, and may thus enhance its transport activity.