Abstract: Objective: To study the mechanism of anti-nonalcoholic fatty liver disease (NAFLD) by didymin based on non-targeted metabolomics and bioinformatics methods. Methods: Eighteen male SD rats were randomly divided into normal group, model group, and didymin group (3 mg/kg), with 6 rats in each group. NAFLD rat model was induced by feeding high-fat diet for 8 weeks. The didymin group was given vanillin every day for 8 weeks, and the normal group and model group were given the same amount of normal saline. Hematoxylin-eosin (HE) staining and Oil Red O staining were used to observe the histopathological changes of liver in each group. The metabolites of rat liver homogenate were detected by ultra-high-performance liquid-mass spectrometry (UPLC-Xevo G2-XS QTof). The differential metabolites were screened based on criteria of P＜0.05 and the change ratio (|FC|)＞2), and the metabolites were classified and enriched. Using total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), free fatty acid (FFA), insulin resistance (IRI) and other NAFLD-related indicators as disease phenotypes, weighted metabolites correlation network analysis (WMCNA) was built and the hub metabolites associated with NAFLD were screened. The intersection of differential metabolites and hub metabolites was analyzed for biomarkers. Results: Compared with the model group, the didymin group could reduce body weight, liver lipid deposition and liver damage in rats. Metabolomics results showed that 126 differential metabolites were selected, of which 50 were up-regulated and 76 were down-regulated (didymin group vs. model group). WMCNA screened 189 hub metabolites, mainly sulfonic acid and its derivatives, fatty acyl compounds, and 25 potential biomarkers were obtained by the intersection of the differential metabolites and hub metabolites in Metaboanlyst dataset. Among them, the differences in the fatty acyl compound Isopropylmalic acid (P＜0.05, FC=-2.81) and the fatty acid-related compounds Alpha-Linoleoylcholine (P＜0.05, FC=-1.39) and 3-trans,5-cis-Octadienoyl-CoA (P＜0.05, FC=-1.78) were statistically significant. Conclusion: Didymin may play a role in improving NAFLD by regulating the metabolic levels of Isopropylmalic acid, Alpha-Linoleoylcholine and 3-trans,5-cis-Octadienoyl-CoA. It provides the preliminary experimental basis for alleviating the disorder of lipid metabolism.