Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl⁻ channels important for anion secretion, we herein performed experiments on Capan-1, a human pancreatic duct cell line, using open-circuit Ussing chamber and gramicidin-perforated patch-clamp techniques. The luminal addition of adenosine increased the negative transepithelial potential difference (V_te) in Capan-1 monolayers with a half-maximal effective concentration value of approximately 10 μM, which corresponded to the value obtained on whole-cell Cl⁻ currents in Capan-1 single cells. The effects of adenosine on V_te, an equivalent short-circuit current (I_sc), and whole-cell Cl⁻ currents were inhibited by CFTRinh-172, a cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel inhibitor. The adenosine A_2B receptor agonist, BAY 60-6583, increased I_sc and whole-cell Cl⁻ currents through CFTR Cl⁻ channels, whereas the A_2A receptor agonist, CGS 21680, had negligible effects. The A_2B receptor antagonist, PSB 603, inhibited the response of I_sc to adenosine. Immunohistochemical analysis showed that the A_2A and A_2B receptors colocalized with Ezrin in the luminal membranes of Capan-1 monolayers and in rat pancreatic ducts. Adenosine elicited the whole-cell Cl⁻ currents in guinea pig duct cells. These results demonstrate that luminal adenosine regulates anion secretion by activating CFTR Cl⁻ channels via adenosine A_2B receptors on the luminal membranes of Capan-1 cells. The present study endorses that purinergic signaling is important in the regulation of pancreatic secretion.