Diabetic cardiomyopathy (DCM) is characterized by cardiac microvascular endothelial cells (CMECs) injury and cardiomyocyte (CM) dysfunction. Exosomes mediated cellular communication between CMECs and CM has emerging roles in the pathogenesis of DCM, but the underlining mechanisms are unclear. Mammalian sterile 20-like kinase 1 (Mst1), a key component in Hippo pathway which participates in regulating organ size, apoptosis and autophagy, is involved in the development of DCM. We generated the endothelial-specific Mst1 transgenic mice (Tg-Mst1) and constructed diabetic model with streptozotocin (STZ). Interestingly, Tg-Mst1 mice suffered from worse cardiac function and aggravated insulin resistance compared with non-transgenic (NTg) diabetic mice. The content of Mst1 protein was increased, while Mst1 mRNA had no significant change in CM isolated from diabetic Tg-Mst1 mice. In vitro, CMECs-derived exosomes were taken up by CM and increased Mst1 protein content which inhibited autophagy, as well as enhanced apoptosis in high glucose (HG) cultured CM as evidenced by immunofluorescence and western blot analysis. In addition, Mst1 inhibited glucose uptake under diabetic condition by disrupting the glucose transporter type 4 (GLUT4) membrane translocation through decreasing the interaction between Daxx and GLUT4, as well as enhancing the association of Mst1 and Daxx. Our study exemplifies pleiotropic effects of Mst1-enriched exosomes released from CMECs on inhibiting autophagy, promoting apoptosis and suppressing the glucose metabolism in CM.