Background: Using a relaxant agent before an endoscopic retrograde cholangiopancreatography (ERCP) might reduce complications. Study Aims: We aimed to investigate the relaxant effects of proton pump inhibitors (PPIs) on sheep sphincter of Oddi (SO) and the mechanisms that might take part in this relaxant effect. Patients and Methods: The sheep SO was mounted in an organ bath filled with Krebs-Ringer bicarbonate solution under 1.5 g tension and the relaxant effects of PPIs were evaluated in the tissues precontracted by carbachol (10(-6) mol/l). The relaxant responses to the PPIs were tested in the presence of various blockers to enlighten the underlying mechanism by the PPIs. Results: The PPIs exerted relaxant responses in a concentration-dependent manner in the sheep SO (P < 0.05). Esomeprazole produced the strongest relaxation. The administration of atropine, indomethacin, L-NAME, methylene blue, clotrimazole, glibenclamide, and 4-aminopyridine into the organ baths did not change the relaxations induced by PPIs in vitro (P> 0.05). On the other hand, Ca+2-activated potassium channel blocker tetraethylammonium (TEA) reduced the relaxation responses created by PPIs (P < 0.05). Conclusions: The present study suggests that PPIs create relaxation on SO partially via Ca+2-activated potassium channels. PPIs, especially esomeprazole, may be beneficial during the ERCP procedure. Further clinical studies are needed to confirm our results.