Background: 1, 25-dihydroxycholecalciferol [1a, 25(OH)2D3], known as calcitriol, is the active metabolite of vitamin D3 that regulates calcium and phosphate homeostasis. Calcitriol plays a key role in the regulation of immune, cardiovascular and nervous systems. Moreover, it regulates cell proliferation and differentiation with even anti-aging activity. Its action is mediated by the vitamin D receptor found in nearly almost all types of cells providing multiple actions on various tissues. Objectives: This study examined whether calcitriol could change the motility of the isolated rabbit ileum segments and investigate its mechanism of action more thoroughly. Methods: Different serial doses of calcitriol were studied on the isolated ileal smooth muscles of the rabbit in the presence and absence of L-NAME (a nitric oxide synthase inhibitor), Ach, CaCl2, and verapamil (a calcium channel blocker). The ileal smooth muscle contractions were recorded using a force transducer. Results: Calcitriol significantly reduced the spontaneous ileal smooth muscle contractions in a dose-dependent manner. Surprisingly, the incubation of ileal tissue with L-NAME (10-6 M) was able to inhibit the relaxation response induced by calcitriol. Moreover, pretreatment with calcitriol (10-6 M) has reduced the Ach-evoked contractile intestinal response. However, calcitriol failed to modify the verapamilinduced relaxation. Conclusion: Calcitriol reduced the spontaneous contractile response of the isolated rabbit ileum. This relaxation response is induced by calcitriol partly mediated by the nitric oxide pathway with the involvement of calcium channels.