Although the role of NF-κB in the pathogenesis of sepsis and septic shock has been extensively studied, little is known about the causative contribution of endothelial-intrinsic NF-κB to these pathological processes. In this study, we used transgenic (TG) mice (on FVB genetic background) that conditionally overexpress the NF-κB inhibitor, mutant I-κBα, selectively on endothelium and their transgene-negative littermates (wild type (WT)) to define the causative role of endothelial-specific NF-κB signaling in septic shock and septic vascular dysfunction. In WT mice, LPS challenge caused systemic hypotension, a significantly blunted vasoconstrictor response to norepinephrine, and an impaired endothelium-dependent vasodilator response to acetylcholine, concomitant with a markedly increased aortic inducible NO synthase expression, significantly elevated plasma and aortic levels of nitrite/nitrate, increased aortic TNF-α expression, and decreased aortic endothelial NO synthase (eNOS) expression. In TG mice whose endothelial NF-κB was selectively blocked, LPS caused significantly less hypotension and no impairments in vasoconstrictor and endothelium-dependent vasodilator responses, associated with significantly reduced aortic inducible NO synthase expression, decreased plasma and aortic levels of nitrite/nitrate, reduced aortic TNF-α expression, and increased aortic eNOS expression. TNF-α knockout mice prevented LPS-induced eNOS down-regulation. WT mice subjected to cecal ligation and puncture showed significant systemic hypotension, which was prevented in TG mice. Our data show that selective blockade of endothelial-intrinsic NF-κB pathway is sufficient to abrogate the cascades of molecular events that lead to septic shock and septic vascular dysfunction, demonstrating a pivotal role of endothelial-specific NF-κB signaling in the pathogenesis of septic shock and septic vascular dysfunction.