Abstract Background Elevated levels of low-density lipoprotein (LDL) cholesterol and its oxidative modification have been described to be involved in the process of atherogenesis. Bilirubin, an antioxidant, prevents oxidative modification of LDL and therefore may protect from atherosclerosis and coronary heart disease (CHD). Impaired brachial artery flow-mediated dilatation (FMD), which means endothelial dysfunction (ED) and carotid intima-media thickness (IMT) are predictors for the development and progression of atherosclerosis. In the present study, FMD and IMT were studied in healthy subjects with lower and higher serum bilirubin concentrations in physiological ranges. Methods Ninety-one healthy subjects between 25 and 45 years of age (47 with lower and 44 with higher serum bilirubin concentrations) were included in this study. Carotid IMT and brachial artery flow-mediated dilatation was measured by means of high-resolution vascular ultrasound. FMD was assessed by establishing reactive hyperemia and endothelium-independent dilatation (EID) was determined by using sublingual isosorbide dinitrate. Results EDD in subjects with lower serum bilirubin concentrations was significantly worse than in those with higher serum bilirubin concentrations (11.6 ± 4.4% versus 7.2 ± 4.7%, respectively, p < 0.0001). EID measurements were not significantly different between the groups (16 ± 5.1% versus 16.8 ± 7%, respectively). In addition, carotid IMT was significantly greater in subjects with lower serum bilirubin concentrations (0.5 ± 0.13 mm versus 0.42 ± 0.07 mm, p < 0.0001). Furthermore, FMD in women with lower serum bilirubin concentrations was significantly lower than in women with higher serum bilirubin concentrations (11.5 ± 4.9% and 17.5 ± 4.7%, respectively, p < 0.001). Accordingly, men with lower serum bilirubin concentrations had significantly lower FMD as compared to hyperbilirubinemic ones (11.7 ± 3.6% versus16.7 ± 4.8%, respectively, p = 0.009). Conversely, carotid IMT was significantly greater in both women and men with lower serum bilirubin concentrations compared to the subjects with elevated serum bilirubin concentrations (0.51 ± 0.08 versus 0.41 ± 0.08, p < 0.001; 0.55 ± 0.12 versus 0.40 ± 0.07, p = 0.002, in women and men, respectively). Conclusion The healthy subjects with lower serum bilirubin concentrations show significant ED and increased carotid IMT, which are predictors for atherosclerosis.