Background: Atrial fibrillation (AF) is closely linked to endothelial dysfunction, yet its mechanisms remain unclear. Extracellular vesicles (EVs), including exosomes, are released by most cell types and mediate intercellular communication. We therefore investigated the role of EVs in endothelial dysfunction associated with AF. Methods: Vascular endothelial function in patients with sinus rhythm (SR), premature ventricular contractions (PVCs), or AF was assessed by peripheral arterial tonometry. Plasma-derived EVs were isolated from these three groups. Conditioned medium was collected from cultured cardiomyocytes (CMs), which were paced either regularly or irregularly at 1 Hz or 10 Hz or were non-paced, and EVs were subsequently isolated from the conditioned media. The isolated EVs were applied to endothelial cells (ECs), and mRNA levels of vasoactive genes were quantified. Results: The reactive hyperemia index (RHI) was significantly lower in patients with AF than in those with SR (RHI values: 1.98 in SR vs. 1.57 in AF, p = 0.049), whereas no significant decrease was observed in patients with PVCs, indicating endothelial dysfunction in AF. The plasma EV concentration was significantly higher in patients with AF than in those with SR. CMs subjected to 10 Hz irregular pacing released more EVs than non-paced cells and cells under 1 Hz regular pacing. When applied to ECs, EVs from patients with AF and from rapidly paced CMs significantly reduced NOS3 mRNA expression in vitro. Conclusions: Circulating EVs are increased in AF and could be associated with an impaired endothelial function in AF.