Abstract Nonalcoholic fatty liver disease (NAFLD) commonly results from excessive dietary fat intake which characterized by obesity and insulin resistance. Wild fruiting body of Antrodia camphorata (AC) was assayed for alleviative effects on NAFLD. An NAFLD animal model was successfully established in male 7-week-old C57BL/6 mice fed with high-fat diet (HFD) for 36weeks. The HFD mice exhibited obese and impaired glucose metabolism. After an induction of NAFLD syndrome, AC was given for one week via gavage. Mice with AC treatment showed lowered (p<0.05) serum TG and TC, lowered (p<0.05) liver TG content, improved (p<0.05) oxidative status (TBARS values and GSH levels), and ameliorated (p<0.05) liver damage (AST, ALT, and LDH values). In addition, AC activated (p<0.05) gene expressions of PPAR-α with its downstream genes in the liver and caused higher (p<0.05) rectal temperature, which showed AC attenuates hepatic lipid accumulation by promoting lipid oxidation and further suggests the role of AC in energy expenditure. Overall, our findings revealed that AC possesses alleviative effect on NAFLD.