Pathologic rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely-used as a model of this event, we characterized gallbladder ion transport in animals fed control chow using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current (Isc, 171 ± 21 µA/cm²) and lumen negative potential difference (-10.1 ± 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, while the residual net ion flux corresponded to Isc. In response to 2 µM forskolin, Isc exceeded 270 µA/cm², and impedance estimates of the apical membrane resistance decreased from 200 cm² to 13 cm². The forskolin-induced Isc was dependent on extracellular HCO₃^- and blocked by serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl- ion substitution had little effect. Serosal 293B and Ba²⁺ reduced Isc consistent with the inhibition of cAMP-dependent K⁺ channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, sodium-bicarbonate cotransporter protein, pNBC1, expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO₃^- entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K⁺ channel activation.