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Background: Impairment of endothelial function is associated with atherosclerosis and atrial fibrillation, and could underlie several types of ischemic stroke. Recently, embolic stroke of undetermined source (ESUS) has attracted much attention as the major cause of cryptogenic stroke.Objective: The purpose of this study was to clarify the endothelial function in patients with ESUS and compare it with those with other major ischemic stroke subtypes and without a history of stroke.Material and Methods: Between 2015 September and 2017 July, we have prospectively enrolled 402 outpatients with any vascular risk factors or with a history of cerebrovascular events, who had undergone brain MRI within a year and had any sign of cerebral vessel disease. Among them, 230 patients underwent flow-mediated vasodilation (FMD) test to evaluate vascular endothelial function. The subject group was classified into No stroke group and five stroke subtype groups; large artery atherosclerosis (LAA), small vessel disease (SVO), cardiogenic embolism (CE), ESUS, and others (Other stroke). Endothelial function was expressed as percent increase of brachial vessel diameter (%FMD) after interruption of blood flow with mechanical compression for five minutes.Results: The age of subjects was 69.5±10.6 years old, and 61% of them were males. The prevalence of hypertension, diabetes, dyslipidemia, chronic kidney disease, atrial fibrillation, and a history of stroke were 69%, 28%, 51%, 20%, 10%, and 57% respectively. The %FMD of total subjects was 5.16±2.60%; %FMD of stroke subtypes were 5.19±0.32% in No stroke (n=100), 5.02±0.60% in LAA (n=18), 5.19±0.37% in SVO (n = 47), 4.97±0.63% in CE (n=16), 3.55±0.50% in ESUS (n=26), 6.55±0.53% in Other stroke (n=23). The %FMD was significantly lower in ESUS than those in No stroke, SVO, Other stroke (p<0.05). No significant difference in %FMD was observed between ESUS group and LAA or CE group.Conclusion: This study clearly showed that vascular endothelial function of patients with ESUS was significantly impaired. Endothelial dysfunction could underlie development of atrial fibrillation, atherosclerotic plaque or thrombus formation in patients with ESUS.