Testicular function and associated testosterone concentration decline with advancing age, and an impaired O₂ supply may contribute, in part, to this reduction. We hypothesized that there would be a reduced microvascular Po₂ (Po₂(m)) in the testes from aged rats, and this reduced Po₂(m) would be associated with impaired vasomotor control in isolated resistance arterioles. In addition, given the positive effect of exercise on microvascular Po₂ and arteriolar function, we further hypothesized that there would be an enhanced Po₂(m) in the testes from aged animals after aerobic exercise training. Testicular Po₂(m) was measured in vivo via phosphorescence quenching in young and aged sedentary (SED) and exercise-trained (ET; 15 m/min treadmill walking, 15-degree incline, 5 days/wk for 10 wk) male Fischer-344 rats. Vasoconstriction to α-adrenergic [norepinephrine (NE) and phenylephrine (PE)] and myogenic stimuli in testicular arterioles was assessed in vitro. In the SED animals, testicular Po₂(m) was reduced by ∼50% with old age (aged SED 11.8 ± 1.9 vs. young SED 22.1 ± 1.1 mmHg; P = 0.0001). Contrary to our hypothesis, exercise training did not alter Po₂(m) in the aged group and reduced testicular Po₂(m) in the young animals, abolishing age-related differences (young ET, 10.0 ± 0.8 vs. aged ET, 10.7 ± 0.9 mmHg; P = 0.37). Vasoconstrictor responsiveness to NE and PE was diminished in aged compared with young (NE: young SED, 58 ± 2 vs. aged SED, 47 ± 2%; P = 0.001) (PE: young SED, 51 ± 3 vs. aged SED, 36 ± 5%; P = 0.008). Exercise training did not alter maximal vasoconstriction to NE in young or aged groups. In summary, advancing age is associated with a reduced testis Po₂(m) and impaired adrenergic vasoconstriction. The diminished testicular microvascular driving pressure of O₂ and associated vascular dysfunction provides mechanistic insight into the old age-related decrease in testicular function, and a reduced Po₂(m) may contribute, in part, to reduced fertility markers after exercise training.