Genipin has been the focus of research as a multifunctional compound for the treatment of pathogenic diseases. However, hepatotoxicity caused by oral genipin raises concerns about its safety. To obtain novel derivatives with low toxicity and efficacy, we synthesized methylgenipin (MG), a new compound, using structural modification, and investigated the safety of MG administration. The results showed that the LD50of oral MG was higher than 1000 mg/kg, no mice died or were poisoned during the experiment in the treatment group, and there was no significant difference in biochemical parameters and liver pathological sections compared with the control. Importantly, MG (100 mg/kg/d) treatment for 7 days reduced alpha-naphthylisothiocyanate (ANIT)-induced increases in liver index, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), and total bilirubin (TBIL) levels. Histopathology demonstrated that MG could treat ANIT-induced cholestasis. In addition, using proteomics to investigate the molecular mechanism of MG in the treatment of a liver injury may be related to enhancing antioxidant function. Kit validation showed that ANIT induced an increase in malondialdehyde (MDA) and a decrease in superoxide dismutase (SOD) and glutathione (GSH) levels, while the MG pretreatments, both of which were significantly reversed to some extent, suggested that MG may alleviate ANIT-induced hepatotoxicity by enhancing endogenous antioxidant enzymes and inhibiting oxidative stress injury. In this study, we demonstrate that the treatment of mice with MG does not cause impaired liver function and provide an investigation of the efficacy of MG against ANIT-induced hepatotoxicity, laying the foundation for the safety evaluation and clinical application of MG.