Excess reactive oxygen species (ROS) produced by the mitochondria are a key mechanism underlying age-related vascular endothelial dysfunction. Aerobic exercise with aging leads to improvements in cardiorespiratory fitness (CRF) and preserves endothelial function in preclinical models via lower mitochondrial-derived ROS (mtROS) however, these effects have yet to be fully translated to humans. Here, we sought to investigate if an underlying mechanism of low CRF secondary to a physically inactive lifestyle contributing to enhanced endothelial function with aging is mediated via lower mtROS in humans from acute (single dose, 80 mg) mitochondrial-specific antioxidant (MitoQ) supplementation. In a double-blind, randomized, placebo-controlled study design 23 (10M/13 F, age 62.1  2.4 y) MA/O adults (≥45 y) were separated into two groups; exercisers (EX, n=11) and non-exercisers (NEX, n=12). All participants completed measures of endothelial function (brachial artery flow-mediated dilation, FMDBA), cardiovascular function (subendocardial viability ratio, SEVR) and arterial stiffness (carotid-femoral pulse wave velocity, cfPWV and common carotid artery [CCA] β-stiffness and elasticity) before and 1 hr. after acute MitoQ intake. An independent two-tailed samples T-test was used to determine descriptive differences between EX and NEX. A two-way repeated measures ANOVA with a Bonferroni ii post-hoc was used to determine the effects of MitoQ and placebo on measures of vascular function and cardiovascular health. Pearson correlations were used to assess the relation between the change () in FMDBA in response to acute MitoQ and CRF. Compared to NEX, EX had lower resting heart rates, body mass index, body fat percentage, arterial stiffness (cfPWV) and higher FMDBA, SEVR and CRF (p<0.05, all). Compared to placebo, MitoQ improved FMDBA in NEX by +2.1% (MitoQ pre: 4.9  0.4 vs post: 7.0  0.4 %, p<0.05) resulting in a +37 relative improvement and +0.06 mm (+34% relative) increase in brachial artery diameter compared to EX (MitoQ pre: 6.8  0.5 vs post: 7.0  0.3 %, p>0.05). Additionally, the improvement (i.e., change) in FMDBA with MitoQ was inversely correlated to CRF (r=-0.66, p<0.05). MitoQ also improved myocardial perfusion (SEVR) in NEX by +10.4% (MitoQ pre: 132.3  6.0 vs post: 142.7  6.6%) compared to EX (MitoQ pre: 176.0  9.9 vs post: 178.3  8.9%). Arterial stiffness, assessed by cfPWV and CCA β-stiffness did not change (p>0.05). These data demonstrate MitoQ acutely improves cardiovascular health in NEX via increases in FMDBA and SEVR. These findings support the notion that a primary mechanism underlying the cardioprotective effects associated with aerobic exercise and higher CRF to preserve endothelial function with aging is mediated, in part, to greater management of mtROS. Therefore, targeting mtROS with MitoQ may represent a novel strategy for enhancing cardiovascular health and preventing future CVD risk with aging in humans.