Hydrogen-rich saline (HS) is reported to be a new therapeutic agent in ischemia-reperfusion (I/R)-induced organ damage. The present study was designed to investigate the beneficial effects of HS against spinal cord I/R injury and its associated mechanisms. Spinal cord ischemia was induced by infrarenal aortic occlusion for 20min in male New Zealand white rabbits. Different doses of HS were intravenously (i.v.) administered at 5min before or after the beginning of reperfusion. Moreover, the roles of mitochondrial ATP-sensitive potassium channels (mitoKATP), oxidative stress, inflammatory cytokines and apoptosis was assessed. Here, we found that I/R-challenged rabbits exhibited significant spinal cord injury characterized by the decreased numbers of normal motor neurons and hind-limb motor dysfunction, which was significantly ameliorated by 5mL/kg and 10mL/kg HS treatment before reperfusion or 10mL/kg HS treatment after reperfusion. However, the protective effects of HS treatment in spinal cord I/R injury were partially abolished by the selective mitoKATP channel blocker 5-hydroxydecanoate (5-HD). Moreover, we showed that the beneficial effects of 10mL/kg HS treatment against spinal cord I/R damage were associated with the decreased levels of oxidative products [8-iso-prostaglandin F2α (8-iso-PGF2α) and malondialdehyde (MDA)] and pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and high-mobility group box 1 (HMGB1)], as well as the increased activities of antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] in serum at 6h, 12h, 24h, 48h and 72h after reperfusion and in spinal cord at 72h after reperfusion. Furthermore, HS treatment (10mL/kg) reduced caspase-3 activity in the spinal cord of this model. Thus, HS may be an effective therapeutic agent for spinal cord I/R injury via activation of mitoKATP channels as well as reduction of oxidative stress, inflammatory cytokines and apoptosis.