Introduction: Pancreatic acini secrete ATP and nucleotide-modifying enzymes that include CD39 and CD73. Adenosine, the end product of ATP, elicited Cl− efflux in pancreatic duct cells that express adenosine receptors (Novak et al. 2008). However, effect of adenosine receptors on anion secretion has not been extensively investigated. Objectives: The present study aimed to determine whether adenosine receptors regulate anion channels in pancreatic duct cells. Methods: Pancreas was obtained from male Wistar rats. Protocols involving handling of animals were approved by the Animal Experimentation Committee, Kansai Medical University. Animals were killed by cervical dislocation. The molecular basis of adenosine receptors was revealed by RT-PCR analysis and immunostaining. We measured equivalent short-circuit current (Isc) in human adenocarcinoma cell line (Capan-1) monolayer. Statistical significance was evaluated using ANOVA. Results: Pancreas expressed adenosine A2A receptor (Adora2a), confirming findings in previous study. Adenosine A2A receptors localized in luminal membrane of rat ducts and Capan-1 monolayer. The luminal addition of adenosine (100 µM) significantly increased Isc in Capan-1 monolayer (n = 25). The effect was consistent with Cl− secretion in epithelia. This increase was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid, niflumic acid, or CFTRinh-172 (n = 5). Conclusion: These results indicated that the adenosine A2A receptors regulate both Ca2+-activated Cl− channels and cystic fibrosis transmembrane conductance regulator in Capan-1 cells. Furthermore, the adenosine A2A receptors may be involved in anion transport in pancreatic ducts.