Increasing serum levels of biliverdin and bilirubin was shown to be beneficial in settings of inflammation. Bilirubin was shown to be protective in LPS-induced lung injury in rats; however, the exact mechanism remains elusive. Here, we investigated whether a single bolus injection of bilirubin would exert anti-inflammatory effects in a mouse model of endotoxemia. Mice were challenged with sublethal doses (2 mg/kg body weight) of LPS, and the effects of intravenously administered bilirubin (40 mg/kg body weight) were assessed. In contrast to control animals, bilirubin-treated animals fully recovered from endotoxin shock within 24 h. Bilirubin treatment improved the clinical score significantly at all time points assessed, attenuated weight loss, and improved LPS-induced anorexia. Furthermore, bilirubin treatment inhibited LPS-induced leukocyte-endothelial interactions and leukocyte accumulation in various tissues. Expression of inflammatory genes, including endothelial adhesion molecules, but also IL-1β and TNF-α, was significantly reduced in bilirubin-treated animals. Moreover, bilirubin inhibited LPS-induced expression of inflammatory genes in isolated cultured aortic endothelial cells and in bone marrow-derived macrophages. These data show that single-dose administration of bilirubin attenuates tissue injury induced by endotoxin, and that bilirubin, in addition to its antioxidant effects, also exerts potent anti-inflammatory activity.