Recent research has indicated that sialic acid, such as free sialic acid (N-acetylneuraminic acid, Neu5Ac) and bound sialic acid (3ʹ-sialyllactose, 3ʹ-SL), can ameliorate disorders associated with glycolipid metabolism, although the underlying mechanisms have yet to be determined. We examined the effects of 3ʹ-SL on glycolipid metabolism in mice fed a high-fat diet. Male C57BL/6J mice were divided into 6 groups: 2 model control groups (normal and high-fat diets) and 4 intervention groups (Neu5Ac, and low-, moderate-, and high-dose 3ʹ-SL). After 8 weeks of continuous gavage intervention, mice in the 3ʹ-SL intervention groups had lower body weight and total fat content; reduced fasting blood glucose, triglycerides, low-density lipoproteins and oxidized-low-density lipoproteins; and increased high-density lipoproteins, but no dosage-dependent of 3ʹ-SL intervention was found, moderate-dose 3ʹ-SL intervention as optimal for further exploration. 3ʹ-SL intervention could increase respiratory exchange ratio, energy expenditure, and amount of exercise performed. 3ʹ-SL increased the colonic abundances of Akkermansia, Lactobacillus, and Bacteroides, and reduced those of Erysipelatoclostridium, Faecalibaculum, and Aldercreutzia. Changes were also observed in colonic metabolites, and liver gene transcript and metabolites, which were mainly enriched in bile secretion, taurine and hypotaurine metabolism, and insulin resistance. Additionally, 3ʹ-SL was observed to regulate genes associated with physiological rhythms, including Clock, Per2, Cry1, and Bhihe41. Collectively, our findings indicate that 3ʹ-SL can contribute to the prevention and control of disorders associated with glucose and lipid metabolism caused by high-fat diets. Compared with Neu5Ac, 3ʹ-SL intervention can more effectively ameliorate intestinal flora disorders, enhance bile acid circulation, increase tissue energy expenditure, and reduce lipid synthesis, thereby promoting lipid-lowering effects mediated via the gut-liver axis, and can also enhance energy metabolism and alleviate disorders of glucolipid metabolism by altering physiological rhythms in high fat-diet mice.