<p>The bile acid-sensitive ion channel (BASIC) is the least understood member of the mammalian epithelial Na⁺ channel/degenerin (ENaC/DEG) superfamily of ion channels, which are involved in a variety of physiological processes. While some members of this superfamily, including BASIC, are inhibited by extracellular Ca²⁺ (Ca²⁺_o), the molecular mechanism underlying Ca²⁺ modulation remains unclear. Here, by determining the structure of human BASIC (hBASIC) in the presence and absence of Ca²⁺ using single-particle cryo-electron microscopy (cryo-EM), we reveal Ca²⁺-dependent conformational changes in the transmembrane domain and β-linkers. Electrophysiological experiments further show that a glutamate residue in the extracellular vestibule of the pore underpins the Ca²⁺-binding site, whose occupancy determines the conformation of the pore and&#xa0;therefore ion flow through the channel. These results reveal the molecular principles governing gating of BASIC and its regulation by Ca²⁺ ions, demonstrating that Ca²⁺ ions modulate BASIC function via changes in protein conformation rather than solely from a pore-block, as proposed for other members of this superfamily.</p>