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Particulate matter 2.5 (PM_2.5) is an environmental pollutant linked with the risk of cardiovascular disease. Aerobic exercise performed in polluted environments may have fewer benefits because of increased PM_2.5 inhalation during exercise. However, the vascular responses to aerobic exercise in high PM_2.5 (HPM_2.5) conditions remain unknown. This study aimed to examine the acute flow-mediated dilation (FMD) response to moderate-intensity treadmill running in HPM_2.5 levels compared to low PM2.5 (LPM_2.5) levels in healthy young males. Treadmill running in both HPM_2.5 and LPM_2.5 levels was completed by nine subjects. Brachial artery FMD was measured before and after the exercise to assess vascular endothelial function. Indoor PM_2.5 concentration was significantly higher in HPM_2.5 than in LPM_2.5 conditions (<i>p</i> < 0.001). Scaled FMD significantly increased after the exercise in LPM_2.5 conditions but not in HPM_2.5 (<i>p</i> = 0.03), and baseline diameter increased only in HPM_2.5 conditions after the exercise (<i>p</i> = 0.001). Baseline diameter and peak diameter were smaller, and time to peak dilation was delayed in HPM_2.5 compared to LPM_2.5 in pre-exercise intervention measurements (<i>p</i> < 0.05). Therefore, acute PM_2.5 exposure can counteract the positive effect of aerobic exercise on vascular endothelial function in young males.