Rhabdomyolysis is characterised by acute kidney injury (AKI) resulting from skeletal muscle injury. Lipid peroxidation‐mediated oxidant injury and pro‐inflammatory cytokine‐mediated inflammatory response play critical roles in the pathogenesis of rhabdomyolysis‐induced AKI. The present study aimed to investigate the short‐term effects of both lipid peroxidation and inflammatory responses on rhabdomyolysis‐induced AKI in a rat model of glycerol‐induced rhabdomyolysis. Rhabdomyolysis was induced by the intramuscular injection of 50% glycerol in saline (10 mL/kg) into the hind limbs of rats. Rats were killed 1 or 3 hours after glycerol injection. Time‐dependent increases in serum biochemical parameters, including blood urea nitrogen, creatinine, lactate dehydrogenase and creatine phosphokinase levels, were observed 1 hour after glycerol injection. In kidneys, glycerol injection resulted in histopathological changes such as renal tubular injury and renal tubular myoglobin deposition. Levels of Nε‐(hexanoyl)lysine‐modified, 4‐hydroxy‐2‐nonenal‐modified, and nitrotyrosine‐modified proteins in rat kidneys were unaltered at 1 hour after glycerol injection, but increased significantly at 3 hours. Increases in renal nitric oxide production and the expression levels of inducible nitric oxide synthase, interleukin‐6 and tumour necrosis factor‐α in the renal parenchyma were observed at 1 hour after glycerol injection and plateaued at 3 hours. Our findings suggest that the pro‐inflammatory cytokine‐mediated inflammatory response may cause rhabdomyolysis‐induced AKI very shortly after glycerol injection, and lipid peroxidation‐mediated oxidant injury may promote the development of these pathophysiological processes.