Cholecystitis is one of the most common gastrointestinal diseases. Inflammation induces activation of proteases that can signal to cells by cleaving protease-activated receptors (PARs) to induce hemostasis, inflammation, pain and repair. However, the distribution of PARs in the gallbladder is unknown, and their effects on gallbladder function have not been fully investigated. We localized immunoreactive PAR₁ and PAR₂ to the epithelium, muscle and serosa of mouse gallbladder. mRNA transcripts corresponding to PAR₁ and PAR₂, but not PAR₄, were detected by RT-PCR and sequencing. Addition of thrombin and PAR1-selective activating peptide (AP, TFLLRN-NH₂) to the serosal surface of mouse gallbladder mounted in an Ussing chamber stimulated an increase in short circuit current (I_sc) in wild-type but not PAR₁^-/- mice. Similarly, serosally-applied trypsin and PAR₂-AP (SLIGRL-NH₂) increased I_sc in wild-type but not PAR₂^-/- mice. Proteases and APs strongly inhibited electrogenic responses to subsequent stimulation with the same agonist, indicating homologous desensitization. Removal of HCO₃^- ions from the serosal buffer reduced responses to thrombin and trypsin by >80%. Agonists of PAR₁ and PAR₂ increase [Ca²⁺]_i in isolated and cultured gallbladder epithelial cells (GBEC). COX-2 inhibitor, meloxicam and an inhibitor of CFTR prevented the stimulatory effect of PAR₁ but not PAR₂. Thus, PAR₁ and PAR₂ are expressed in the epithelium of the mouse gallbladder, and serosally applied proteases cause a HCO₃^- secretion. The effects of PAR₁ but not PAR₂ depend on generation of prostaglandins and activation of CFTR. These mechanisms may influence fluid and electrolyte secretion of the inflamed gallbladder, when proteases are generated.