Bilirubin, a byproduct of heme catabolism, has been shown to be an effective lipid-soluble antioxidant in vitro. Bilirubin is able to inhibit free radical chain reactions and protects against oxidant-induced damage in vitro and ex vivo. However, direct evidence for bilirubin's antioxidant effects in vivo remains limited. As bilirubin is formed from biliverdin by biliverdin reductase, we generated global biliverdin reductase-a gene knockout (Bvra–/–) mice to assess the contribution of bilirubin as an endogenous antioxidant. Bvra–/– mice appear normal and are born at the expected Mendelian ratio from Bvra+/– x Bvra+/– matings. Compared with corresponding littermate Bvra+/+ and Bvra+/– animals, Bvra–/– mice have green gall bladders and their plasma concentrations of biliverdin and bilirubin are approximately 25-fold higher and 100-fold lower, respectively. Naïve Bvra–/– and Bvra+/+ mice have comparable plasma lipid profiles and low-molecular weight antioxidants, i.e., ascorbic acid, α-tocopherol and ubiquinol-9. Compared with wild-type littermates, however, plasma from Bvra–/– mice contains higher concentrations of cholesteryl ester hydroperoxides (CE-OOH), and their peroxiredoxin 2 (Prx2) in erythrocytes is more oxidized as assessed by the extent of Prx2 dimerization. These data show that Bvra–/– mice experience higher oxidative stress in blood, implying that plasma bilirubin attenuates endogenous oxidative stress.