<p>Exercise training improves endothelial function and reduces vascular inflammation. However, whether aerobic exercise training-induced secretion of irisin, a myokine cleaved from fibronectin type III domain-containing protein 5 (<i>FNDC5</i>), is associated with endothelial nitric oxide synthase (eNOS)-mediated improvements in endothelial function and inflammation, thereby affecting atherosclerosis, remains unclear. In this study, apolipoprotein E-deficient (ApoE^−/−) mice, an atherosclerotic disease model, were assigned to sedentary (ApoE^−/− SED) or aerobic exercise training (voluntary wheel running for 16 weeks; ApoE^−/− AT) groups, with sedentary wild-type (C57BL/6J) mice (WT-SED) as healthy controls. Atherosclerotic lesion area, aortic interleukin-6 and tumor necrosis factor-α protein expression, and intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels were significantly higher, whereas acetylcholine-induced vasorelaxation and aortic eNOS expression were significantly lower in ApoE^−/− SED mice than in WT-SED mice. Aerobic exercise training improved these parameters in ApoE^−/− mice. Soleus <i>FNDC5</i> mRNA expression and plasma irisin levels were reduced in ApoE^−/− SED mice but increased in ApoE^−/− AT mice. Circulating irisin levels positively correlated with soleus <i>FNDC5</i> mRNA and aortic eNOS expression. These findings suggest that aerobic exercise-induced irisin secretion is associated with improved endothelial function and reduced vascular inflammation, possibly involving eNOS, which suppresses atherosclerosis.</p>