Vascular oxidative stress contributes to endothelial dysfunction. Aerobic exercise training improves vascular function. The purpose of this study was to test the hypothesis that exercise training would improve the balance of anti- to pro-oxidant enzymes, and reduce markers of oxidative stress in aortic endothelial cells (AEC). Female Yucatan miniature pigs either remained sedentary (SED) or were exercise trained (EX) for 16-19 weeks. EX increased AEC SOD-1 protein levels, and Cu/Zn SOD activity of whole aorta compared to SED. Protein levels of other anti-oxidant enzymes (SOD-2, catalase) were not affected by EX. Protein levels of p67^phox, a subunit of the pro-oxidant enzyme NAD(P)H oxidase, were reduced in EX vs SED AEC. These EX adaptations were associated with lower AEC malondialdehyde levels, and decreased phosphorylation of ERK-1/2. Endothelial nitric oxide synthase protein, protein nitrotyrosine content and HO-1 protein were not different in EX vs SED. We conclude that chronic aerobic exercise training influenced both anti- and pro-oxidant enzymes and decreased indices of oxidative stress in AEC. These adaptations may contribute to improved endothelial function with exercise training.