Objective To investigate the effects of Osteoking on hyperglycemia and regulating gut microbiota in db/ db mice. Methods Wildtype mice were used as the control group, and db/ db mice were randomly divided into model and Osteoking groups. After intragastric administration for 12 weeks, fasting blood glucose and serum glycosylated hemoglobin and insulin levels were measured, changes in intestinal microflora were determined, and functional pathways related to intestinal microflora in mice were predicted by 16S rDNA sequencing. Results Compared with the model group, Osteoking decreased fasting blood glucose (P< 0. 01), serum glycosylated hemoglobin (P< 0. 01), and the insulin resistance index (P<0. 01), and increased insulin content (P<0. 01) in db/ db mice. Osteoking increased the abundance of beneficial intestinal microflora and decreased the abundance of harmful bacteria. Moreover, the abundance of Marvinbryantia was increased. Osteoking alleviated the decrease in metabolism of D-arginine and D-ornithine, sphingolipid, and galactose metabolism (P<0. 05) and inhibited lysine degradation, the sulfur relay system, and propanoate metabolism (P< 0. 05). Conclusions Osteoking has hypoglycemic properties and improves the intestinal microflora imbalance in db/ db mice.