Objective? To investigate the effect and mechanism of homocysteine (Hcy) regulated Forkhead box O3a (FOXO3a) -mediated mitochondrial damage in metabolic associated fatty liver disease (MAFLD).? Methods?Six weeks old Cbs+/- mice (n=12) were randomly divided into 2 groups: normal diet for 12 weeks (ND，n=6) and high methionine diet for 12 weeks (HMD，n=6).Cell Counting Kit-8 Kit was used to detect the inhibition ratio of hepatocytes treated with different concentrations of Hcy (0, 50, 100, 150 μmol/L). NCTC1469 mice normal hepatocytes were divided into Control group (Control) and Hcy intervention group (Hcy, 100 μmol/L Hcy)，NC siRNA-transfected control group (si-NC) and FOXO3a siRNA-transfected group （si-FOXO3a），NC siRNA-transfected with Hcy intervention group （Hcy+si-NC） and FOXO3a siRNA-transfected with Hcy intervention group （Hcy+si-FOXO3a）. HE staining was used to observe liver tissue injury. Oil red O was used to observe the distribution and accumulation degree of lipid droplets in hepatocytes. Total cholesterol (TC) and triglycerides (TG) were used to analyze the contents of lipid metabolites in cells. Western blot was used to detect the differences in FOXO3a protein expression in mouse liver tissues (ND and HMD) and liver cells (Control and Hcy ). ROS was used to observe the degree of oxidative stress in cells. JC-1 was used to detect the mitochondrial membrane potential. Mito-tracker was used to observe the morphological changes of mitochondria. qRT-PCR was used to detect the change of mtDNA expression in each group.? Results? Compared with ND, the structure of liver tissue in HMD was disordered, the hepatocytes were enlarged, the cytoplasm was loose and light stained, and a large number of vacuoles and lipid droplets were observed in the liver cells. Compared with the Control, the number of oil red O positive lipid droplets in the Hcy was increased, and the levels of TC and TG were increased. Compared with the ND and the Control, the expression of FOXO3a protein in the HMD and the Hcy was significantly increased. Compared with the Hcy+si-FOXO3a, the Hcy+si-FOXO3a had decreased intracellular ROS level, significantly decreased JC-1 monomer, decreased mitochondrial fragmentation, decreased number of oil red O-positive lipid droplets, and decreased TC and TG levels. ?Conclusion? FOXO3a plays an important promoting role in lipid metabolic disorders of MAFLD caused by Hcy by regulating mitochondrial damage.