Purpose: The beneficial effects of exercise training on the cardiovascular system are well known. Because our knowledge of exercise-induced vascular function is still limited, we aimed to uncover the molecular mechanisms conditioning the improved vascular relaxation in muscular arteries. Methods: Male Wistar–Kyoto rats with the same ability to run on a treadmill after maximal exercise tests were allocated to the following two groups: trained (Tr) (treadmill, 50%–60% of maximal capacity, 5 d·wk−1) and untrained (UnTr). After 13 wk, the femoral arteries were harvested and used for functional, structural, and molecular analyses. Results: Acetylcholine (ACh)-induced relaxation and nitric oxide (NO) production were enhanced in arteries from Tr rats compared with UnTr rats. Tr arteries exhibited reduced microRNA (miRNA)-124a expression (whose target is caveolin-1), increased the density of caveolae aligned along the sarcolemma and reduced ACh-induced relaxation in the presence of methyl-β-cyclodextrin, which disrupts caveolae. Higher endothelial NO synthase (eNOS) expression with lower miRNA-155 expression and the posttranslational modification of eNOS (phosphorylation of stimulatory Ser1177 and dephosphorylation of inhibitory Thr495) by the PI3-kinase/Akt1/2/3 pathway also contributed to the higher NO production induced by exercise training. Furthermore, increased Cu/Zn- and extracellular-superoxide dismutase expression and enhanced effects of their pharmacological scavenger activity on the ACh-induced response were observed in Tr arteries. Conclusions: The results of the present study provide a molecular basis for exercise-induced NO bioavailability in healthy femoral arteries. Increased caveolae domain and eNOS expression/activity in Tr arteries are associated with downregulation of miRNA-124a and -155, as well as are involved with higher antioxidant defense, subsequently inducing a favorable endothelium-dependent milieu in Tr arteries. [ABSTRACT FROM AUTHOR]