The human IFI16 gene is an interferon-inducible gene implicated in the regulation of endothelial cell proliferation and tube morphogenesis. Immunohistochemical analysis has demonstrated that this gene is highly expressed in endothelial cells in addition to hematopoietic tissues. In this study, gene array analysis of human umbilical vein endothelial cells (HUVEC) overexpressing IFI16 revealed an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. Consistent with these observations, IFI16 triggered expression of adhesion molecules such as ICAM-1 and E-selectin or chemokines such as IL-8 or MCP-1. Treatment of cells with small interfering RNA (shRNA) targeting IFI16 significantly inhibited ICAM-1 induction by IFN- demonstrating that IFI16 is required for proinflammatory gene stimulation. Moreover, functional analysis of ICAM-1 promoter by deletion- or sitespecific mutation demonstrated that NF-B is the main mediator of IFI16 driven gene induction. NF-B activation appears to be triggered by IFI16 through a novel mechanism involving suppression of IBa mRNA and protein expression. Further support to this finding comes from the observation that IFI16 targeting with specific shRNA downregulates NF-B binding activity to its cognate DNA, and inhibits ICAM-1 expression induced by IFN-. Using transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation we demonstrate indeed that activation of the NF-B response is mediated by IFI16-induced block of Sp1-like factor recruitment to the promoter of the IBa gene, encoding the main NF-B inhibitor. Activation of NF-B accompanied by induction of proinflammatory molecules was also observed when IBa expression was downregulated by specific siRNA, resulting in an outcome similar to that observed with IFI16 overexpression. Taken together, these data implicate IFI16 as a novel regulator of endothelial proinflammatory activity and provide new insights into the physiological functions of the IFN-inducible gene IFI16.