Endothelium has long been considered both a source and a target of systemic inflammation. However, to what extent endothelial activation contributes to systemic inflammation remains unclear. This study addresses the relative contribution of endothelial activation to systemic inflammation and multiple organ dysfunction and injury (MOD/I) in an E. coli peritonitis model of sepsis. We prevented endothelial activation using transgenic (TG) mice that conditionally overexpress a mutant I‐κBα, a NF‐κB inhibitor, selectively on endothelium. TG mice and their transgene negative littermates (WT) were injected with saline or E. coli (10⁸ CFU per mouse). At 7 h after E. coli infection, markers of systemic inflammation, endothelial activation, and MOD/I were assessed. WT‐E. coli mice showed significantly increased serum levels of TNF‐α, IL‐1β, IFN‐γ, IL‐6, KC, and MCP‐1; tissue levels of TNF‐α, IL‐6, KC, MCP‐1, ICAM‐1, and VCAM‐1; endothelial leakage index in heart, lungs, liver, and kidney; significantly increased serum levels of AST, ALT, BUN, and creatinine; and increased mortality. Blockade of NF‐κB‐mediated endothelial activation in TG mice had no effects on serum levels of TNF‐α, IL‐1β, IFN‐γ, IL‐6, KC, and MCP‐1 (markers of systemic inflammation), and tissue levels of TNF‐α, IL‐6, KC, and MCP‐1, but significantly reduced tissue levels of ICAM‐1 and VCAM‐1 (markers of endothelial inflammation and activation) in those four organs. TG‐E. coli mice displayed reversed endothelial leakage index; reduced serum levels of AST, ALT, BUN, and creatinine; and improved survival. Our data demonstrate that endothelial NF‐κB‐driven inflammatory response contributes minimally to systemic inflammation, but plays a pivotal role in septic MOD/I, suggesting that endothelium is mainly a target rather than a source of systemic inflammation. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.