An impaired decline in blood pressure at rest is typical in people with diabetes, reflects endothelial dysfunction, and increases the risk of end-organ damage. Here we report that microRNA-204 (miR-204) promotes endothelial dysfunction and impairment in blood pressure decline during inactivity. We show that db/db mice overexpress miR-204 in the aorta, and its absence rescues endothelial dysfunction and impaired blood pressure decline during inactivity despite obesity. The vascular miR-204 is sensitive to microbiota, and microbial suppression reversibly decreases aortic miR-204 and improves endothelial function, while the endothelial function of mice lacking miR-204 remained indifferent to the microbial alterations. We also show that the circulating miR-122 regulates vascular miR-204 as miR-122 inhibition decreases miR-204 in endothelial cells and aorta. This study establishes that miR-204 impairs endothelial function, promotes impairment in blood pressure decline during rest, and opens avenues for miR-204 inhibition strategies against vascular dysfunction.