Extracellular vesicles (EVs) have been described as novel biomarkers and bioactivators in vascular dysfunction in hypertension. However, the mechanism(s) by which EVs affect vascular function is unknown. To examine the effects of EVs on endothelial-dependent vasodilation (acetylcholine), we isolated circulating EVs from platelet-poor plasma using a low centrifugation speed (17 000 g ) and mesenteric resistance arteries from 12-week-old normotensive WKYs (Wistar-Kyoto rats) and SHRs (spontaneously hypertensive rats). Arteries were cannulated on a pressure myograph, and EVs were added to the vessel lumen and circulating bath. We found that circulating EVs from normotensive WKY reduced vasodilation of normotensive WKY arteries but had no effect on hypertensive SHR arteries. In contrast, EVs from hypertensive SHR failed to reduce vasodilation of arteries from both WKY and SHR. The restraining effect on vasodilation by EVs from normotensive WKY may be mediated by inhibition of eNOS (endothelial NO synthase), as addition of L-nitro-arginine methyl ester did not provide any additive effect. Moreover, circulating EVs from normotensive 6-week-old SHR—an age where SHRs have not yet developed hypertension—had similar restraining effect on vasodilation. In addition, delipidation of EVs did not alter the restraining effect of EVs from WKY but did restore the restraining effect of EVs from SHR. Finally, EVs from normotensive humans also restrained vasodilation of normotensive mouse arteries—an effect not observed in EVs from hypertensive humans. Taken together, our data support a vasoactive role of EVs that is altered in hypertension.