Objective: To investigate the mechanism of lactoferrin(LF) regulating the disorder of glucose and lipid metabolism in mice induced by high-fat diet. Methods: Thirty SPF grade male C57BL/6 mice were randomly divided into a control group (Group K， normal diet)， model group (Group M， high-fat diet drinking pure water)， and LF treatment group (Group Y2， high-fat diet drinking 2% lactoferrin water). The mice were fed continuously for 12 weeks. Body weight changes of the mice were recorded weekly. Visceral fat percentage was measured at week 12 by taking peri-epididymal abdominal adipose tissue. Blood glucose and lipid levels were analyzed using commercial enzymatic assay kits. Insulin levels were measured using ELISA. 16S rRNA sequencing was used to measure the intestinal flora of the mice. Short-chain fatty acids were detected by gas chromatography-mass spectrometry. Results: After 12 weeks of lactoferrin intervention， the visceral fat percentage of mice in Y2 group decreased by 31.05% compared with mice in M group. Blood glucose (5.92 mmol/L)， insulin (19.60 mmol/L), total cholesterol (3.17 mmol/L)， triglyceride (0.28 mmol/L)， low density lipoprotein (1.84 mmol/L) levels were all decreased and the differences were significant compared with group M (P<0.05). The high density lipoprotein level (1.88 mmol/L) was significantly increased compared with that of group M (P<0.05). Lactoferrin intervention decreased the ratio of Firmicutes/Bacteroidetes and increased the relative abundance of Bacteroidetes， decreased the relative abundance of Colidextribacter and Deferribacteres， regulated the abnormal metabolism of short-chain fatty acids， and controlled the accumulation of fat. Conclusions: Lactoferrin regulates the accumulation of fat by regulating the structure of intestinal flora， and improves the disorder of glucose and lipid metabolism in high-fat diet mice.