Introduction: DCM is a complex disease with many possible causes and multifactorial pathophysiology, but its underlying mechanisms are poorly understood, and no specific therapeutic guideline has yet been established. Increasing evidence suggests that coronary microvascular dysfunction (CMD) might be one of the primary mechanisms in DCM development. CMD is characterized by impaired endothelial-dependent vasodilation, but detailed mechanisms have yet to be elucidated. Sirtuin 6 (Sirt6) has been implicated in obesity, insulin resistance, type 2 diabetes mellitus, and cardiovascular diseases. Sirt6 protects EC from premature senescence, oxidative stress, and atherosclerosis by sustaining high eNOS levels and preserving cell replication. How Sirt6 regulates coronary microvascular function remains to be determined.Methods: Inducible Sirt6 global knockout, endothelial-specific knockout, and wild-type (WT) mice were fed a diet high in fat and sugar (HFHS), and blood lipid and glucose were measured. Coronary arteries were isolated, and endothelial-dependent vasodilation (EDD) was assessed using myography (DMT). Echocardiography and treadmill exercise exertion tests were performed to evaluate cardiac function. Myocardial blood flow (MBF) was measured by doppler. Cardiac fibrosis was detected using trichrome staining, and molecular pathways were elucidated via gene and protein analysis.Results: Our preliminary data show that Sirt6 was downregulated in diabetes. The EDD of coronary arterioles Sirt6 global knockout and endothelial-specific knockout treated with HFHS was impaired, and the mediator of coronary vasodilation switched from NO to H2O2 in the Sirt6 knockout mice. Compared to the WT mice, myocardial blood flow was decreased, ejection fraction (EF) was not changed, the running distance was reduced, and E/E’ ratio was increased in Sirt6 knockout mice. The Sirt6 targeted proteins were identified.Conclusions: Our suggest Sirt6 regulated coronary microvascular function through endothelial cells, and ablation of Sirt6 in endothelial cells caused CMD and diastolic dysfunction. Further genetic profiling will elucidate the pathways and mechanisms converging with Sirt6 to regulate microvascular function.