BACKGROUND This study aims to investigate the association of exposure to ambient air pollution during pregnancy with cord blood concentrations of surrogate markers of endothelial dysfunction. METHODS This population-based cohort was conducted from March 2014 to March 2015 among 250 mother-neonate pairs in urban areas of Isfahan, the second large and air-polluted city in Iran. We analyzed the association between the ambient carbon monoxide (CO), ozone (O3), nitrogen dioxide (NO2), sulfur dioxide (SO2), particular matter 10 (PM10), and air quality index (AQI) with cord blood levels of endothelin-1, vascular adhesion molecule (VCAM), and intercellular adhesion molecule (ICAM). Multiple regression analysis was conducted after adjustment for potential confounding factors and covariates. The regression coefficient (beta), standard error of the estimate (SE), and 95% confidence intervals for each regression coefficient (95% CI) are reported. RESULTS Data of 233 mother-neonate pairs were complete, and included in the analysis. Multiple regression analyses showed that AQI, CO and O3 had significant correlation with cord blood ICAM-1 [Beta (SE), 95%CI: 2.93 (0.72), 1.33,5.54; 2.28(1.44), 1.56,5.12; and 2.02(0.01), 1.03,2.04, respectively] as well as with VCAM-1 [2.78(0.91), 1.69,4.57; 2.47(1.47), 1.43,5.37; and 2.01(0.01),1.07,2.04, respectively]. AQI, PM10, and SO2 were significantly associated with Endothelin-1 concentrations [Beta (SE), 95%CI: 10.16(5.08),7.61,14.28; 9.70(3.46), 2.88,16.52; and 1.07(0.02), 1.03,2.11, respectively]. CONCLUSIONS The significant associations of air pollutants with markers of endothelial dysfunction during fetal period may provide another evidence on the adverse health effects of air pollutants on early stages of atherosclerosis from fetal period. Our findings underscore the importance of considering environmental factors in primordial prevention of chronic diseases.