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[Display omitted] • Gastrodigenin rhamno-pyranoside (GR) was separated from the seeds of Moringa seeds. • GR inhibited lipids accumulation and enhanced antioxidant capacity in L02 cells. • GR decreased serum lipid and showed hepatoprotection in NAFLD model mice. • Anti-NAFLD effect of GR was mediated by the AMPK/mTOR/SREBP-1 pathway in L02 cells. Moringa seed extract containing 1-O-(4-hydroxy-methylphenyl)-α- l -rhamno-pyranoside (GR) has been reported to ameliorate CCl4 induced hyperlipidemia in the liver. However, it is unclear whether GR has any therapeutic effect on NAFLD. This study aimed to determine the hypolipidemic and concomitant hepatoprotective potential of GR. The structure and purity of GR were assessed and characterized using high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). The NAFLD model was established based on the L02 cells in vitro. After GR intervention, intracellular fat deposition and reactive oxygen content were significantly reduced, GR could up-regulation of AMPK and PPARα and the down-regulation of mTOR and SREBP-1, which play a key role in liver lipid homeostasis. In vivo experiments revealed that GR intervention significantly decreased serum fat content, inhibited liver injury, and increased antioxidant mechanism in mice fed with high fat diet. Hence, GR demonstrated promising hypolipidemic and hepatoprotective activities, serving as a potential candidate for NAFLD therapy. [ABSTRACT FROM AUTHOR]