Polygonatum sibiricum is a medicinal and homologous plant grown in China, which is commercially available without the provision of medical prescription. Saponin is one of the biologically active components of Polygonatum sibiricum. This study aimed to extract saponin from Polygonatum sibiricum (PSS) and to investigate its hypoglycemic effect and effect on gut micorbiota in diabetic mice. Through single factor and orthogonal experiments, the extraction conditions of saponin were optimized, and the content of saponin in the extract was 3.07 ± 0.02 mg g-1. During the experiment evaluating the hypoglycemic ability, it was found that PSS could inhibit the activity of α-amylase and α-glucosidase at first. Then, HepG2 was used to observe the effect of the saponin on insulin resistance. The results showed that PSS could significantly improve the state of insulin resistance in IR-HepG2 cells, increase the glucose consumption and intracellular glycogen content of cells, and the activity of hexokinase (0.455 ± 0.007 μmol min-1 g-1) and pyruvate kinase (0.174 ± 0.038 U per g prot). Finally, it was found that PSS could alleviate the symptoms of polyphagia and polydipsia in diabetic mice, regulate the gut microbiota of diabetic mice, increase the number of probiotics, and reduce the number of harmful bacteria. In summary, PSS may play an important role in regulating diabetes and can be developed as a promising natural material for diabetes prevention and treatment.