BACKGROUND: Rhabdomyolysis is a leading cause of acute kidney injury. The pathophysiological process involves oxidative stress and inflammation. Hydrogen-rich saline (HRS) is an antioxidant and anti-inflammatory. This study explored the protective effect of pretreatment with HRS on the development of glycerol-induced rhabdomyolysis acute kidney injury. MATERIALS AND METHODS: Forty-eight rats were randomly divided into four equal groups. Group 1 served as the control, group 2 was given 50% glycerol (10 mL/kg, intramuscular), group 3 was given glycerol after 7 d pretreatment with high dose HRS (10 mL/kg/d, intraperitoneal), and group 4 was given glycerol after 7 d pretreatment with low dose HRS (5 mL/kg/d, intraperitoneal). Renal health was monitored by serum creatinine (Cr), urea, and histologic analysis; rhabdomyolysis was monitored by creatine kinase (CK) levels; and oxidative stress was monitored by kidney tissue reactive oxygen species (ROS), malondialdehyde, 8-hydroxydeoxyguanosine (8-OH-dG), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) levels. Inflammation was monitored by interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) evaluation. RESULTS: Glycerol administration resulted in an increase in the mean histologic damage score, serum Cr, urea and CK, kidney tissue ROS, malondialdehyde, 8-OH-dG, GSH-PX, IL-6, and TNF-α, and a decrease in kidney tissue superoxide dismutase activity. All these factors were significantly improved by both doses of HRS, but the mean histologic damage score, urea, Cr, CK, ROS, 8-OH-dG, GSH-PX, IL-6, and TNF-α for the high dose HRS treatment group were even lower. CONCLUSIONS: Pretreatment by HRS ameliorated renal dysfunction in glycerol-induced rhabdomyolysis by inhibiting oxidative stress and the inflammatory response.