The objectives of this study were to determine whether treatment with the hydrogen sulfide (H2S) donor NaHS (NaHS‐PC) 24 hrs prior to ischemia/reperfusion (I/R) would prevent postischemic mitochondrial dysfunction in rat intestinal mucosa and, if so, whether calcium‐activated, large conductance potassium (BKca) channels were involved in this protective effect. BKCa expression in intestinal mucosa was detected by immunohistochemistry and western blotting. I/R was induced by 45‐min occlusion of the superior mesenteric artery (SMA) followed by 60‐min reperfusion in rats preconditioned with NaHS (0.8 mg/Kg, ip) 24 hrs earlier. Mitochondrial function was assessed by measuring the mitochondrial membrane potential (JC‐1), mitochondrial dehydrogenase function (MTT), and cytochrome c release. The protective effect of NaHS‐PC on postischemic mitochondrial function was abolished by coincident treatment with a BKca channel inhibitor, paxilline (2.5 mg/Kg, ip). In addition, preconditioning with a BKca channel activator (NS‐1619, 1 mg/Kg, ip) in lieu of NaHS‐PC was also effective in preventing I/R‐induced mitochondria dysfunction. Moreover, the protective effects of NS‐1619 preconditioning were abolished by coincident treatment with paxilline. Our data demonstrate that NaHS‐PC prevents postischemic mitochondrial dysfunction by a BKca channel‐dependent mechanism.