Bestrophin calcium-activated chloride channels (CaCCs) regulate the flow of chloride and other monovalent anions across cellular membranes in response to intracellular calcium (Ca2+) levels. Mutations in bestrophin 1 (BEST1) cause certain eye diseases. Here we present X-ray structures of chicken BEST1-Fab complexes, at 2.85 angstrom resolution, with permeant anions and Ca2+. Representing, to our knowledge, the first structure of a CaCC, the eukaryotic BEST1 channel, which recapitulates CaCC function in liposomes, is formed from a pentameric assembly of subunits. Ca2+ binds to the channel's large cytosolic region. A single ion pore, approximately 95 angstrom in length, is located along the central axis and contains at least 15 binding sites for anions. A hydrophobic neck within the pore probably forms the gate. Phenylalanine residues within it may coordinate permeating anions via anion-pi interactions. Conformational changes observed near the 'Ca2+ clasp' hint at the mechanism of Ca2+-dependent gating. Disease-causing mutations are prevalent within the gating apparatus.