Methotrexate (MTX) is effective for the therapy of cancer, lupus erythematosus, rheumatoid arthritis, psoriasis and other diseases. However, the hepatotoxicity of MTX often leads to withdrawal and discontinuation of drugs, and then limits its clinical application. Monotropein (MON), an iridoid glycosides distributing in many edible and medicinal plants, has been exhibited to prevent liver and kedney injury induced by chemicals and related diseases. The current study aimed to assess the potential beneficial effects of MON in mitigating MTX-induced liver injury together with delineating the implicated mechanisms. A MTX-induced liver injury model in mice and BRL 3A cells were developed to explore the hepatoprotection of MON. GEO and WGCNA analysis were conducted to predict the possible mechanism of MTX on liver injury. The results demonstrated that MON can decrease the serum levels of AST and ALT, increase the activities of antioxidant SOD and GSH, and improve the morphological alteration of liver tissue, and reduce the accumulation of lipids in hepatocytes and liver tissue of mice treated with MTX. Moreover, our results also exhibited that MON can activate autophagy as assessed by autophagosome formation, expression of protein related with autophagy, and inhibit ferroptosis as evaluated by mitochondrial integrity, Fe²⁺level and key protein expression changes, and CQ (inhibitor of autophagy) and RSL-3 (the activator of ferroptosis) reversed the effects of MON on autophagy and ferroptosis of hepatocytes injured by MTX. Therefore, MON attenuates liver injury of MTX by activating autophagy and inhibiting ferroptosis. These findings provide new insights into MON as a potential healthy ingredient in functional food.