This study aimed to identify and elucidate the mechanism underlying the protective effect of tricin-enriched Zizania latifolia (Z. latifolia) extract (ETZL) against free fatty acid (FFA)-induced lipid accumulation in vitro and non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet and fructose diet (HFD/F) in vivo. ETZL treatment significantly lowered body weight gain and decreased adipose tissue, lipid, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels in HFD/F-fed mice. ETZL acted on phosphorylated acetyl-CoA carboxylase (ACC) and anti-peroxisome proliferator-activated receptor α (PPARα) by activating the adenosine monophosphate-activated protein kinase (AMPK) pathway and inhibiting sterol regulatory element-binding proteins-1 (SREBP)/fatty acid synthase (FAS) signaling to inhibit de novo adipogenesis and increase fatty acid oxidation. In addition, treatment with ETZL increased nuclear factor erythroid-2-related factor 2 (Nrf2) levels to activate the antioxidant pathway. FFA-induced oxidative stress and fatty acid accumulation in HepG2 cells confirmed the improvement in fat accumulation through the AMPK and Nrf2 pathway activities of ETZL. These results suggest that ETZL ameliorates NAFLD by regulating lipid metabolism and defending against oxidative stress via AMPK-dependent pathways.