Abstract: The imbalance of glucose and lipid metabolism severely endangers human health. We aimed to investigate the effect of 3, 4-dihydroxyphenylacetic acid on the body weight, serum lipids, and glucose tolerance of mice exposed to a high-fat diet. Forty-five male C57BL/6 mice were randomly divided into three groups:normal control group, high-fat diet control group (HFD group), and 3, 4-dihydroxyphenylacetic acid group (high-fat diet+0.05% 3, 4-dihydroxyphenylacetic acid). During the experimental period, body weight, food intake, and water intake were measured weekly. After 10 weeks of treatment, Lee's index, organ weight to body weight ratios, perirenal fat and epididymal fat coefficient, serum lipid parameters (triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol), and glucose tolerance were examined. 3, 4-dihydroxyphenylacetic acid not only significantly reduced body weight gain (HFD group, (9.50±1.16) g; 3, 4-dihydroxyphenylacetic acid group, (7.54±1.22) g) and Lee's index, but also markedly lowered the perirenal fat and epididymal fat coefficients. 3, 4-dihydroxyphenylacetic acid also significantly reduced liver index (HFD group, (3.45±0.09)%; 3, 4-dihydroxyphenylacetic acid group, (3.30±0.25)%). Furthermore, compared with the HFD group, the serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels of mice in the 3, 4-dihydroxyphenylacetic acid group were significantly decreased by 20.90%, 26.00%, and 45.09%, respectively. Surprisingly, the levels of serum total cholesterol and triglyceride in the 3, 4-dihydroxyphenylacetic acid group basically reached the level of the normal group, indicating that 3, 4-dihydroxyphenylacetic acid significantly reduced the level of blood lipids in HFD-fed mice. Moreover, the fasting blood glucose and AUC values in the 3, 4-dihydroxyphenylacetic acid group were significantly lower than those in the HFD group. The blood glucose values of 3, 4-dihydroxyphenylacetic acid group were close to those of normal group at 120 min, indicating that 3, 4-dihydroxyphenylacetic acid significantly improved glucose tolerance. Therefore, 3, 4-dihydroxyphenylacetic acid showed the biological activity in regulating the disorders of glucose and lipid metabolism in obese mice.