We tested the hypothesis that oxidative stress contributes to reductions in left ventricular diastolic (LV) function in estrogen-deficient postmenopausal women, related in part to reduced nitric oxide (NO) bioavailability.LV diastolic function – recorded using transthoracic echocardiography and determined as the peak early (E) to late (A) mitral inflow velocity ratio and the E to peak early (e’) mitral annular velocity ratio – and brachial artery flow mediated dilation (FMD), a biomarker of NO bioavailability, were measured during acute systemic infusions of saline (control) and ascorbic acid (experimental model to decrease oxidative stress) in healthy premenopausal women (N=14, 18–40 years) and postmenopausal women (N=23, 45–75 years).The E/A ratio was lower (1.16[1.06–1.33] vs 1.65[1.5–2.3]; median[interquartile range]) and the E/e’ ratio was elevated (8.8[7.6–9.9] vs. 6.6[5.5–7.3]) in postmenopausal compared with premenopausal women, indicating reduced LV diastolic function. E/A and E/e’ were correlated with FMD (r=0.54 and r=−0.59, respectively, both P<0.01). Ascorbic acid infusion improved both FMD (5.4±2.0% to 7.8±2.6%) and E/e’ (to 8.1[7.2–9.7], P=0.01) in postmenopausal women but not in premenopausal women. Ascorbic acid did not change E/A in either group.The current study provides evidence that oxidative stress contributes to reduced LV diastolic function in estrogen-deficient postmenopausal women, possibly by reducing the availability of NO.