Amikacin is one of the aminoglycoside antibiotics widely used in intensive care units for pediatric and adult patients, especially for serious infections such as life-threatening Gram-negative infections. This study investigated whether misoprostol, which has antioxidant and antiapoptotic properties, had a beneficial effect on liver damage caused by amikacin administration in rats. Misoprostol treatment increased serum high-density lipoprotein levels and significantly decreased alkaline phosphatase, alanine transaminase, aspartate transaminase, total cholesterol, low-density lipoprotein, direct bilirubin, total bilirubin, and triglyceride levels. Cytochrome P450 2B1 and Cytochrome P450 2B2 gene expressions induced by amikacin treatment were ameliorated by misoprostol. Liver weights increased by the phospholipidosis effect of amikacin were improved by misoprostol treatment. A decrease in malondialdehyde levels and an increase in glutathione activities were detected in the amikacin+misoprostol group compared to the amikacin group. Oxidative damage caused by amikacin was ameliorated by misoprostol treatment due to its antiapoptotic and antioxidative effects. Treatment with misoprostol significantly reduced Caspase 3 and Tumor Necrosis Factor-alpha levels compared to amikacin. Our results showed that misoprostol could serve as a new therapeutic target to prevent amikacin-induced hepatotoxicity by inhibiting reactive oxygen species generation and modulating apoptosis.