Abstract: 【Objective】 To investigate the efficacy of ureidosuccinic acid（UA）in regulating lipid and energy metabolism in vitro and in vivo，and to clarify its effects on non-alcoholic fatty liver disease and the potential mechanisms，which could provide theoretical basis for the application of UA in the prevention and treatment of non-alcoholic fatty liver di-sease. 【Method】 Using oleic acid/palmitic acid（OA/PA）-treated HepG2 cells as a cellular model，cellular oxidative stress， mitochondrial ATP levels and mitochondrial fluorescence were assessed to validate the role of UA in alleviating non-alcoholic fatty liver disease at the cellular level. Additionally，C57BL/6J mice which were fed with high-fat diet（HFD） were served as the biological model. Body weight，liver weight，triglyceride（TG）content and total cholesterol（TC） content were measured，and liver tissue sections were prepared. Real-time fluorescence quantitative PCR was employed to evaluate the expression of fatty liver related genes，further confirming the function of UA in alleviating non-alcoholic fatty liver disease in vivo. Moreover，reverse target finding and molecular docking techniques were applied to identify potential targets of UA for mitigating fatty liver. 【Result】 100 μmol/L UA effectively reduced TG accumulation in induced HepG2 cells，extremely significantly inhibited fat deposition gene（Pparγ）expression（P<0.01，the same below），extremely significantly reduced ROS level，extremely significantly increased CAT activity，significantly increased GSH level and SOD activity（P<0.05，the same below），and significantly increased cellular mitochondrial DNA（mtDNA） transcription levels and ATP contents. In the mouse test，100 mg/kg UA reduced the liver weight and body weight of mice fed with HFD，significantly inhibited the accumulation of TG and TC in the liver，and significantly down-regulated the expression of the Pparγ and significantly up-regulated the expression of the mitochondrial biosynthesis promoting gene Pgc1α. Histone lysine demethylase 4B（KDM4B） might be a potential target for UA to alleviate non-alcoholic fatty liver disease，with a binding energy of -78.2345 kJ/mol and a binding interaction energy of -77.9353 kJ/mol. 【Conclusion】 UA improves non-alcoholic fatty liver disease induced by HFD in mice by reducing oxidative stress related to obesity， promoting lipid catabolism and metabolism，and improving energy metabolism，as well as interacting with KDM4B protein.