Introduction: Acute kidney injury (AKI) is a severe complication of rhabdomyolysis (RM), where skeletal muscle injury leads to the release of cell contents into the bloodstream, ultimately obstructing renal tubules. This results in renal dysfunction due to increased oxidative stress, inflammation, and apoptosis. Glycerol (10 mL/kg) injection is one of the most common methods to induce experimental AKI; but 10 mL/kg dosage seems to be harmful to rats because we have observed some side effects. This study was designed to evaluate the effects of isoflurane pretreatment in the glycerol model of acute kidney injury, but at first we tried to find a better dosage of glycerol to induce AKI less harmful. Methods: 28 male Wistar rats were used in our investigation. We first studied to find the most effective dosage of glycerol for AKI induction in three groups (5, 6.25, and 10 mL/kg), and accordingly 6.25 mL/kg was selected. Secondly, we investigated isoflurane (1.5%, 20 minutes) pretreatment effects on glycerol-induced AKI by estimating blood urea nitrogen (BUN), creatinine (Cr), Bax/Bcl-2 proteins ratio (Bcl-2-associated X/B-cell lymphoma 2), malondialdehyde (MDA), superoxide dismutase (SOD), and histological changes in renal tissues. Results: The results showed that isoflurane pretreatment suppressed oxidative stress and apoptosis, and therefore was able to improve renal function. Conclusion: Isoflurane pretreatment might be protective against rhabdomyolysis-induced AKI because of its anti-oxidant and anti-apoptotic activities.