Background The internal anal sphincter is a specialized continuation of the circular smooth muscle layer of the rectum. It has been found that internal anal sphincter tonus is significantly increased in the majority of patients with anal fissure. Adrenomedullin has a relaxant effect on smooth muscle in different kinds of tissue. In this in vitro study, we aimed to investigate whether adrenomedullin had a relaxant effect on isolated rabbit internal anal sphincter, and the possible involvement of nitric oxide, prostaglandins, and K+ channels. Methods. Internal anal sphincter smooth muscle strips obtained from New Zealand rabbits were studied in vitro. The effects of adrenomedullin were investigated in isolated strips of rabbit internal anal sphincter smooth muscle precontracted with carbachol alone, and in the presence of indomethacin (a cyclooxygenase inhibitor), N-omega-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthetase inhibitor), and K+ channel blockers. Results. Adrenomedullin caused relaxation of the isolated precontracted rabbit internal anal sphincter strips in a concentration-dependent manner. The response of the internal anal sphincter was not affected by L-NAME, indomethacin, and the K+ channel blockers. Conclusions. This relaxant effect of adrenomedullin might lead to novel clinical applications of adrenomedullin for anorectal disorders with increased internal anal sphincter tonus, such as anal fissure.