Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease globally. The progression of NAFLD is complex and associated with inflammation, oxidative stress, autophagy, endoplasmic reticulum stress, and insulin resistance. Mangiferin, a natural C-glucosyl xanthone, has been reported to show multiple biological activities. The aim of this study was to investigate the therapeutic effect of mangiferin on NAFLD and the underlying molecular mechanism. We established a mouse model of NAFLD using a high-fat diet (HFD), and injected the mice with different doses of mangiferin (15, 30, and 60mg/kg, intraperitoneal) for 12 weeks. Liver tissue was assessed to evaluate changes in inflammatory responses, autophagy, and glycolipid metabolism. We found that mangiferin decreased body weight, as well as the levels of triglycerides and total cholesterol in plasma and the liver. It also increased glucose tolerance in HFD-fed mice. In addition, mangiferin decreased inflammatory responses by inhibiting the activities of nuclear factor kappa B and c-Jun N-terminal kinase, regulated autophagy via the AMP-activated protein kinase/mechanistic target of rapamycin signaling pathway, and improved glycolipid metabolism via modulation of the insulin receptor substrate/phosphoinositide 3-kinase/protein kinase B signaling pathway. This study demonstrated that mangiferin significantly ameliorates NAFLD development in HFD-fed mice by inhibiting inflammatory responses, activating autophagy, and improving glycolipid metabolism.