Diabetes is a chronic metabolic disease characterized by elevated blood glucose levels due to insulin resistance and β-cell dysfunction. This study aims to examine the effects of polysaccharides from adlay seeds (PAS) on hyperglycemia and gut microbiota in streptozocin (STZ)-induced diabetic mice. The administration of PAS in diabetic mice caused a significant decrease in the glucose level and serum levels of glycosylated hemoglobin (HbA1c). Similarly, PAS also showed decreased total cholesterol (TC) and triglyceride (TG) concentrations. Furthermore, a significant increase in the concentrations of glucagon-like peptide 1 (GLP-1) was observed. Unexpectedly, PAS reduced the concentrations of anti-amyloid beta (Aβ 1-42 ) protein. Also, histopathological examination showed that PAS contributed to the reduction of STZ-lesioned pancreatic cells. Metformin treatment significantly reduced the diversity of the gut microbiota, while PAS treatment altered the diversity and composition of the microbiota. Collectively, our findings demonstrate that the hypoglycemic effects of PAS in type-2 diabetic mice (T2D) may be associated with the regulation of the intestinal microbiota and its metabolic pathways. The hypoglycemic effects of PAS in type-2 diabetic mice (T2D) may be associated with the regulation of the intestinal microbiota and its metabolic pathways.