To investigate the effect of baicalein in improving non-alcoholic fatty liver disease caused by high fat-induced oxidative damage in mice. Male C57BL/6J mice weighing 18-20 g were randomly divided into 4 groups: normal control group (C, 10% fat for energy), high-fat group (H, 60% fat for energy), high-fat + scutellaria baicalein group (H+B, baicalein: 400 mg·kg(-1)·bw(-1)), and baicalein control group (B, baicalein: 400 mg·kg(-1)·bw(-1)). After 12 weeks, mice were sacrificed, and the tissue samples were collected. Liver pathological changes were observed by hematoxylin and eosin staining. Mitochondrial morphology was examined by ultramicropathology. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and mitochondrial membrane potential (MMP) changing levels in the liver were determined by kit. Sestrin2 and protein carbonylation (PCOS) levels were detected by Western blotting. Small interfering RNA (siRNA) was used to knock-down the Sestrin2 protein expression in HepG2 cells. Intramyocellular lipid changes in HepG2 cells was detected by fluorescent dye BODIPY493/503. One way ANOVA was used LSD pairwise comparison method was used to test the statistical difference. Compared with the normal control group, high-fat fed caused significant fatty degeneration, decreased GSH and SOD levels ( ​​< 0.05), increased MDA and protein carbonylation levels, and increased Sestrin2 expression ( < 0.05) in mice. Mitochondrial shape changes, swelling, lack of cristae, and MMP was down-regulated by 33.3% ( = 13.456, ​​< 0.001). Baicalein intervention had effectively inhibited hepatic steatosis and oxidative damage caused by high-fat fed, and further up-regulated Sestrin2 expression, MMP ( = 10.104, ​​< 0.001), and significantly alleviated liver damage in mice. Sestrin2 expression knock-down had further increased the intracellular lipid deposition and PCOs expression ( ​​< 0.05), and reduced baicalein ability to antagonize lipid deposition and antioxidant capacity in Hep2 cells. Baicalein alleviate non-alcoholic fatty liver by regulating Sestrin2 expression and high-fat fed-induced liver oxidative damage.