To define the roles of endothelial-intrinsic nuclear factor κB (NF-κB) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-κBαmt) that conditionally overexpress a degradation-resistant form of the NF-κB inhibitor I-κBα (I-κBαmt) selectively on vascular endothelium. The EC-rtTA/I-κBαmt mice had no basal, but a relatively high level of doxycycline-inducible, I-κBαmt expression. I-κBαmt 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-κBαmt mice showed endothelial-selective blockade of NF-κB 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-κBαmt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-κB 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.