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To study the mechanism of lactoferrin (LF) regulating metabolic disorders in nutritionally obese mice through intestinal microflora. Twenty-one male C57BL/6 mice were randomly divided into 3 groups: control group, model group and LF treatment group. The mice in control group were fed with maintenance diet and drank freely. The mice in model group were fed with high fat diet and drank freely. The mice in LF treatment group were fed with high fat diet and drinking water containing 2% LF freely. Body weight was recorded every week. Visceral fat ratio was measured at week 12. Blood glucose and serum lipid level were detected by automatic biochemical analyzer. The gut microbiota of mice was examined using 16 s rRNA sequencing method. LF treatment significantly reduced the levels of visceral adipose ratio, blood glucose, triglyceride, total cholesterol and low-density lipoprotein cholesterol (LDL-C) in high-fat diet mice (p < 0.05). It can be seen that drinking water with 2% LF had a significant impact on metabolic disorders. At the same time, the Firmicutes/Bacteroidetes ratio(F/B) of LF treated mice was decreased. The abundance of Deferribacteres, Oscillibacter, Butyricicoccus, Acinetobacter and Mucispirillum in LF treatment group were significantly decreased, and the abundance of Dubosiella was significantly increased (p < 0.05). In the LF-treated group, the expression levels of glucose metabolism genes in gut microbiota were increased, and the expression levels of pyruvate metabolism genes were decreased. It can be seen that metabolic disorders were related to intestinal flora. In conclusion, LF regulates metabolic disorders by regulating intestinal flora.