Background: Acetaminophen (APAP) is a readily available and safe painkiller. However, its overdose is the most common cause of acute liver injury (ALI). Many predisposing factors contribute to susceptibility to APAP-induced ALI. Non-alcoholic fatty liver disease (NAFLD), the major cause of chronic liver disease, is considered an important predictor of APAP-induced ALI, although the exact mechanism controversial. In this study, we aimed to elucidate the effects of NAFLD on APAP-induced ALI.Methods: Two groups of mice, normal chow (NC) diet-fed and fast food (FF) diet-fed mice for 14 weeks, were further divided into two subgroups: intraperitoneally injected with either saline (NC-S and FF-S groups) or APAP (NC-A and FF-A groups). Biochemical tests, histological analysis, quantitative PCR, and western blotting were conducted.Results: Alanine aminotransferase (ALT) level (199.0 ± 39.0 vs. 63.8 ± 7.4 IU/L, p Conclusions: Our results showed that while the FF diet clearly induced non-alcoholic steatohepatitis and metabolic syndrome, NAFLD also attenuates APAP-induced ALI by inducing anti-inflammatory molecules such as PPAR-γ.