In order to investigate the protective effect of the ethanol extract of Lonicera macranthoides on oxidative stress induced by D-galactose in mouse embryonic fibroblasts(MEF) and its potential mechanism, the active substances in the ethanol extract of Lonicera macranthoides were detected by extensive target metabolome sequencing and high performance liquid chromatography. The MEF cells were treated with 20 mg/mL D-galactose to establish a cell aging model. According to experimental groups, the MEF cells of the control group(0 mg/mL D-galactose), model group(20 mg/mL D-galactose), Lonicera macranthoides ethanol extract group(100, 500, 1000 μg/mL ethanol extract of Lonicera macranthoides added with 20 mg/mL D-galactose) and positive control group(50 μg/mL Panax notoginseng saponin added with 20 mg/mL D-galactose) had been grew for 48 hours, observed the number of cells with ordinary light microscope, detected the inhibition rate of cells with CCK-8 method, detected the activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA) with biochemical method, detected the level of mitochondrial membrane potential(MMP) and mitochondrial reactive oxygen species(mtROS) with flow cytometry, and detected the expression of p53, p16. Besides, the expression of mitochondrial complex gene, Bcl-2, Caspase-3, and the expression of mitochondrial complex protein, Bcl-2, Caspase-3 were detected by Western Blot. The results showed that the ethanol extract of Lonicera macranthoides contained phenolic acids, flavonoids, organic acids, terpenoids and other active substances. Compared with the control group, the inhibition rate of cells in the model group was significantly increased andcompared with the model group, the ethanol extract group of Lonicera macranthoides and the positive control group effectively reduced the cell inhibition rate(P＜0.05), significantly increased the number of cells(P＜0.05, P＜0.01) and SOD activity, decreased the content of MDA(P＜0.05, P＜0.01), increased MMP, decreased the level of mtROS(P＜0.01), effectively reduced the protein levels of p53, p16, Caspase-3, and increased the protein levels of Bcl-2 and NDUFV1(P＜0.05). It showed that in MEF cells, the ethanol extract of Lonicera macranthoides can resist the oxidative stress induced by D-galactose by increasing the concentration of mitochondrial complex I protein and slowing down cell apoptosis