To define the roles of endothelial-intrinsic nuclear factor kappaB (NF-kappaB) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-kappaB alpha mt) that conditionally overexpress a degradation-resistant form of the NF-kappaB inhibitor I-kappaB alpha (I-kappaB alpha mt) selectively on vascular endothelium. The EC-rtTA/I-kappaB alpha mt mice had no basal, but a relatively high level of doxycycline-inducible, I-kappaB alpha mt expression. I-kappaB alpha mt expression was detected in endothelial cells, but not in fibroblasts, macrophages, and whole blood cells, confirming that transgene expression was restricted to the endothelium. When subjected to endotoxemia, EC-rtTA/I-kappaB alpha mt mice showed endothelial-selective blockade of NF-kappaB activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infiltration into multiple organs, decreased endothelial permeability, ameliorated multiple organ injury, reduced systemic hypotension, and abrogated intravascular coagulation. When subjected to cecal ligation and puncture-induced sepsis, the TG mice had less severe multiple organ injury and improved survival compared with wild-type (WT) mice. WT and EC-rtTA/I-kappaB alpha mt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-kappaB activity is an essential mediator of septic multiple organ inflammation and injury but plays little role in the host defense response to eradicate invading pathogenic bacteria.