Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and affects millions of people worldwide. Despite the increasing prevalence of NAFLD, the exact molecular/cellular mechanisms remain obscure and effective therapeutic strategies are still limited. It is well-accepted that free fatty acid (FFA)-induced lipotoxicity plays a pivotal role in the pathogenesis of NAFLD. Inhibition of FFA-associated hepatic toxicity represents a potential therapeutic strategy. Glycyrrhizin (GL), the major bioactive component of licorice root extract, has a variety of pharmacological properties including anti-inflammatory, antioxidant, and immune-modulating activities. GL has been used to treat hepatitis to reduce liver inflammation and hepatic injury; however, the mechanism underlying the antihepatic injury property of GL is still poorly understood. In this report, we provide evidence that 18 β-glycyrrhetinic acid (GA), the biologically active metabolite of GL, prevented FFA-induced lipid accumulation and cell apoptosis inin vitroHepG2 (human liver cell line) NAFLD models. GA also prevented high fat diet (HFD)-induced hepatic lipotoxicity and liver injury inin vivorat NAFLD models. GA was found to stabilize lysosomal membranes, inhibit cathepsin B expression and enzyme activity, inhibit mitochondrial cytochrome c release, and reduce FFA-induced oxidative stress. These characteristics may represent major cellular mechanisms, which account for its protective effects on FFA/HFD-induced hepatic lipotoxicity.Conclusion:GA significantly reduced FFA/HFD-induced hepatic lipotoxicity by stabilizing the integrity of lysosomes and mitochondria and inhibiting cathepsin B expression and enzyme activity.