Objectives/Goals: Heart disease is a leading cause of death in Alabama. Non-Hispanic Black (NHB) women demonstrate greater arterial stiffness than Non-Hispanic White (NHW) women. We explored whether cardiorespiratory fitness (VO2max) accounts for population differences in arterial stiffness among postmenopausal women. Methods/Study Population: We analyzed data from 57 postmenopausal women (65% NHB; age: 62±8 years; BMI: 28±4 kg/m2; blood pressure: 136±17/80±9 mmHg). VO2max was measured using maximal treadmill testing (Modified Bruce Protocol) and arterial stiffness (pulse wave velocity, PWV) via SphygmoCor XCEL. We used bias-corrected bootstrapped mediation models (5,000 resamples) to estimate the indirect effect of ancestry on PWV via VO2max, adjusting for age, body mass index, lived experience, adverse childhood exposures, and neighborhood deprivation. Given the small sample size, these analyses are exploratory. Results/Anticipated Results: NHB women had significantly lower VO2max than NHW women (18.0±3.7 vs. 23.8±4.5 mL•kg−1•min−1). VO2max was inversely associated with PWV (B = -0.10, SE = 0.04, p = 0.02, 95% CI = −0.19 to −0.01). After adjustment, ancestry was not directly associated with PWV. Neighborhood deprivation (B = 0.16, SE = 0.07, p = 0.02, 95% CI = 0.03 to 0.29) and lived experience (B = 0.10, SE = 0.04, p = 0.03, 95% CI = 0.01 to 0.19) were associated with higher PWV, even after adjusting for VO2max and risk factors. In bootstrapped mediation models, the indirect effect of ancestry on PWV through VO2max was significant (B = 0.35, SE = 0.19, 95% BCa CI = 0.03 to 0.78), consistent with partial mediation. Discussion/Significance of Impact: Cardiorespiratory fitness appears to partly account for population differences in arterial stiffness, but this cross-sectional analysis with a modest sample is exploratory. Targeting both fitness and upstream socioeconomic factors may be needed to reduce differences in women’s vascular health.