R3588 --> Introduction: Coronary microvascular dysfunction is particularly common among diabetes and overlaps with impaired endothelial‐dependent vasodilation. These impairments are seen in early stages of the disease, but detailed mechanisms have yet to be elucidated. Nitric oxide (NO) is the major endothelial‐dependent mediator of vasodilation in the healthy coronary circulation, but this mediator changes to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a risk factor for CAD, we hypothesized that a similar switch would occur. Methods: Coronary arteries were isolated, and endothelial‐dependent vasodilation was assessed using myography (DMT). Reactive oxygen species (ROS) in coronary endothelial cells were measured using electron paramagnetic resonance (EPR). Quantitative polymerase chain reaction (qPCR) was performed for gene expression analysis. In vivo myocardial blood flow (MBF) was measured by contrast echocardiography during increased cardiac work achieved through the administration of norepinephrine. Results: Our ex vivo and in vivo data show nitric oxide synthase inhibitor (L‐NAME) inhibited vasodilation in wild type (WT) mice, but the H2O2 scavenger (PEG‐catalase) had no effect. In contrast, vasodilation in diabetic mice was blunted by PEG‐catalase, but not L‐NAME. This suggested that the mediator of coronary vasodilation switched from NO to H2O2 in diabetes. Importantly, we found that microRNA‐21 (miR‐21) was upregulated in diabetes and the deficiency modulated the mediator switch from NO to H2O2 in diabetic mice. Conclusions: The switch in the mediator of coronary vasodilation from NO to H2O2 contributed to microvascular dysfunction in diabetes and miR‐21 regulated this switch. Further genetic profiling will elucidate the pathways and mechanisms converging with miR‐21 to regulate microvascular function in diabetes. This is the first mouse model that recapitulates the switch in mediator of coronary vasodilation from NO to H2O2 seen in CAD patients. [ABSTRACT FROM AUTHOR]