Background and Purpose: Diosmetin (Dios), a flavonoid compound with multiple pharmacological activities. However, fewer studies have reported its effects on diabetes. Here, we address the effect of Dios on glucose metabolism and gut microbiota in KK-Ay diabetic mice. Experimental Approach: Wild type C57BL/6J mice or diabetic KK-Ay mice were treated with vehicle or Dios for one month. The liver RNA-Seq was used to reveal the key signaling pathway interfered with Dios. The liver 16S rRNA gene sequencing was used to reveal the effects of Dios on gut microbiota. The experiment of C. glu transplantation was used to reveal the relationship between Dios and C. glu on glucose metabolism. Key Results: Dios treatment significantly decreased blood glucose and increased serum insulin concentrations. Transcriptome sequencing analysis found that the underlining mechanism of diosmetin on T2DM by regulating signal pathways of glucose metabolism, which was proved by up-regulating IRS/PI3K/AKT signaling pathway to promote glycogen synthesis and GLUT4 translocation. Besides, Dios treatment reshaped the unbalanced gut microbiota by suppressing the ratio of Firmicutes/Bacteroidetes and markedly increasing the richness of C. glu. Moreover, Treatment with C. glu and Dios together can markedly ameliorate glucose metabolism by up-regulating IRS/PI3K/AKT signaling pathway to promoting glycogen synthesis and GLUT4 translocation. Conclusions and Implications: Dios treatment remarkably ameliorated glucose metabolism in KK-Ay diabetic mice by the regulation of C. glu on IRS/PI3K/AKT signaling pathway and reshaped the unbalanced gut microbiota. Our study provided evidence for the application of Dios to the treatment of T2DM.