Dioscin, one natural product, has active effect against non-alcoholic fatty liver disease (NAFLD) in our previous work. However, the pharmacological data are insufficient and the mechanisms have not been reported. Thus, this study aims to comprehensively investigate the effects and molecular mechanisms of dioscin against NAFLD. The primary cultured hepatocytes, AML-12 and HepG-2 cells were treated with palmic acid (PA) after dioscin treatment. The mice and rats were induced by high fat diet to establish the in vivo models of NAFLD. Dioscin obviously alleviated liver lipid accumulation symptoms and improved the levels of serum and hepatic biochemical parameters in vitro and in vivo. Further investigations revealed that dioscin significantly attenuated lipid metabolism via adjusting SIRT1/AMPK signal pathway to regulate the expression levels of SREBP-1c, CPT, FAS, SCD, FoxO1 and ATGL. In addition, suppression of SIRT1 by Nicotinamide or abrogation of AMPK by Compound C eliminated the inhibitory effects of dioscin on lipid metabolism. Therefore, our findings further demonstrated that dioscin markedly prevented NAFLD through adjusting lipid metabolism via SIRT1/AMPK signal pathway, which should be developed as a new candidate for NAFLD.