Aging-induced arterial stiffening is reduced by aerobic exercise training, and elevated production of nitric oxide (NO) participates in this effect. Adropin is a regulator of endothelial NO synthase and NO release, and circulating adropin level decreases with age. However, the effect of habitual aerobic exercise on circulating adropin levels in healthy middle-aged and older adults remains unclear. We sought to determine whether serum adropin level is associated with exercise training-induced changes in arterial stiffness. First, in a cross-sectional study, we investigated the association between serum adropin level and both arterial stiffness and cardiorespiratory fitness in 80 healthy middle-aged and older subjects (65.6 ± 0.9 years old). Second, in an intervention study, we examined the effects of 8-week aerobic exercise training on serum adropin level and arterial stiffness in 40 healthy middle-aged and older subjects (67.3 ± 1.0 years) divided into two groups: aerobic exercise training and sedentary controls. In the cross-sectional study, serum adropin level was negatively correlated with carotid β-stiffness (r = -0.437, P < 0.001) and positively correlated with plasma NOx level (r = 0.493, P < 0.001) and cardiorespiratory fitness (r = 0.457, P < 0.001). Serum adropin levels were elevated after the 8-week aerobic exercise training intervention, and training-induced changes in serum adropin level were correlated with training-induced changes in carotid β-stiffness (r = -0.399, P < 0.05) and plasma NOx level (r = 0.623, P < 0.001). Thus, the increase in adropin may participate in the exercise-induced reduction of arterial stiffness.