Objective:To investigate the effects of Osteoking on hyperglycemia and regulating gut microbiota in db/db mice. Methods:The wild mice were used as the cntrol group and db/db mice were randomly into model group and Osteoking group. After intragastric administration for 12 weeks, Fasting blood glucose, serum glycosylated hemoglobin and insulin levels were measured , the changes of intestinal microflora were determined and functional pathways related to intestinal microflora in mice were predicted by 16S rDNA sequencing technology. Results:Compared with model group, Osteoking decreased fasting blood glucose (P < 0.01), serum glycosylated hemoglobin (P < 0.01), insulin resistance index (P < 0.01), and increased insulin content (P < 0.01) in db/db mice. Osteoking increased the abundance of beneficial bacteria of intestinal microflora, while decreased the abundance of harmful bacteria; the abundance of Marvinbryantia was increased. Osteoking alleviated the decrease of metabolism of D-arginine and D-ornithine, sphingolipid and galactose metabolism( P < 0.05), while inhibited lysine degradation, sulfur relay system and propanoate metabolism( P < 0.05). Conclusion:Osteoking has the effects of hypoglycemic properties and improving the intestinal microflora imbalance in db/db mice.