Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. Nur77 is highly expressed in vascular endothelial cells (ECs) and plays a role in the regulation of cell proliferation and angiogenesis; its role in vascular inflammation, however, remains unknown. Treatment of human umbilical vein ECs (HUVECs) with tumor necrosis factor (TNF)-alpha substantially increased the transcription and protein expression of Nur77 in a dose and time-dependent manner, as determined by Northern blot and Western blot analysis. Adenovirus mediated overexpression of Nur77 markedly increased the intracellular levels of IkappaBalpha by approximately 4-fold, whereas overexpression of dominant negative Nur77 (DN-Nur77), which lacks its transactivation domain, had no effect on IkappaBalpha expression, suggesting that Nur77 is an important transcriptional factor in controlling IkappaBalpha expression in ECs. Furthermore, overexpression of Nur77 significantly increased IkappaBalpha promoter activity via directly binding to a Nur77 response element in the IkappaBalpha promoter. Importantly, overexpression of Nur77, but not DN-Nur77, protected ECs against the TNF-alpha- and interleukin-1beta-induced endothelial activation, as characterized by attenuation in the nuclear factor kappaB activation, expression of adhesion molecules ICAM-1 and VCAM-1, and monocytic adherence to ECs. These results indicate that Nur77 negatively regulates the TNF-alpha- and interleukin-1beta-induced vascular EC activation by transcriptionally upregulation of IkappaBalpha expression.