Objective To investigate the effects and mechanism of homocysteine (Hcy)-regulated FOXO3amediated mitochondrial damage in metabolic dysfunction-associated fatty liver disease (MAFLD). Methods Sixweek-old Cbs ^{+ / -} mice (n= 12) were divided randomly into two groups and fed a normal diet group (ND group) or a high-methionine diet group (HMD group), respectively, for 12 weeks (n = 6 mice per group). The inhibition rate of hepatocytes after treatment with different concentrations(0、50、100、150 μmol / L) of Hcy was detected by the Cell Counting Kit-8 Kit. NCTC1469 normal mouse hepatocytes were divided into the following groups: Control group, Hcy intervention group (Hcy group, 100 μmol / L Hcy), negative control (NC) small interfering ( si) RNA-transfected group ( si-NC group), FOXO3a siRNA-transfected group ( si-FOXO3a group), NC siRNA-transfected with Hcy intervention group ( Hcy + si-NC group), and FOXO3a siRNA-transfected with Hcy intervention group ( Hcy + siFOXO3a group). Liver tissue injury was observed by HE staining and the distribution and accumulation of lipid droplets in hepatocytes was detected by Oil Red O staining. Total cholesterol ( TC) and triglycerides ( TG) were analyzed to indicate the lipid metabolite contents of the cells. FOXO3a protein expression in liver tissues (ND group and HMD group) and liver cells (Control group and Hcy group) were detected by Western blot. Reactive oxygen species (ROS) were measured to indicate the degree of oxidative stress in cells. Mitochondrial membrane potential was detected using JC-1 and morphological changes in mitochondria were observed using Mito-tracker. Changes in mtDNA expression were detected by quantitative reverse transcription-polymerase chain reaction. Results Liver tissue structure was disordered in the HMD group compared with the ND group, hepatocytes were enlarged, the cytoplasm was loose and lightly stained, and a large number of vacuoles and lipid droplets were observed in the liver cells. The number of Oil Red O-positive lipid droplets was increased in the Hcy group compared with the Control group, and TC and TG levels were increased( P<0. 001). Expression levels of FOXO3a protein were significantly increased in the HMD group and Hcy group compared with the ND group and Control group(P<0. 05). Intracellular ROS levels were decreased in the Hcy+si-FOXO3a group compared with the Hcy+si-NC group, and JC-1 monomer,mitochondrial fragmentation, mtDNA expression level, the number of Oil Red O-positive lipid droplets, and TC level and TG level were also all decreased(P<0. 01, P<0. 001). Conclusions FOXO3a plays an important promoting role in lipid metabolic disorders of MAFLD caused by Hcy by regulating mitochondrial damage.