Heme oxygenase-1 (HO-1) is highly protective in various pathophysiological states such as cardiovascular and neurodegenerative diseases. HO-1-derived bilirubin is an efficient scavenger of reactive oxygen and nitrogen species (RONS). It remains to determine whether conversion of biliverdin to bilirubin is an essential step for HO-1-conferred protection of endothelial cells. RONS scavenging activities of biliverdin versus bilirubin were assessed by different RONS generating systems and detection techniques. We also silenced the biliverdin reductase (BVR) or HO-1 gene in cultured primary human endothelial cells (HUVECs) and measured the effect on RONS formation upon stimulation with lipopolysaccharide (LPS). In addition, effects of bilirubin and biliverdin on expression of GTP-cyclohydrolase were assessed in an endothelial cell line (EA.hy 926). HO-1- and BVR-silenced cells have increased levels of oxidative stress and bilirubin but not biliverdin increased expression of the protective protein GTP-cyclohydrolase. Moreover, protection by hemin-induced HO-1 expression or biliverdin-triggered bilirubin formation was impaired upon silencing of the HO-1 or BVR gene, respectively. Since bilirubin significantly scavenged RONS but chronic treatment was even more protective our observations support direct and indirect antioxidant properties of BVR and bilirubin and an important role for BVR and bilirubin in HO-1 conferred protection of endothelial cells.