Dengue virus is the most prevalent arboviral infection especially in tropical and subtropical regions, affecting millions of people annually. Life-threatening complications of dengue virus infection include potentially hemorrhage and shock. The pathogenic mechanisms are not well understood. However, the factors that likely contribute to these manifestations include the activation of monocytes/macrophages and the consequent release of a cascade of vasoactive cytokines. The highly conserved non-structural protein NS1 is secreted from infected cells as a hexameric, lipid-associated species, and circulates at remarkably high levels in the blood of severe dengue patients. We hypothesize that NS1 might contribute to the disease progression by triggering excessive innate immune responses leading to the production of vasoactive cytokines and promoting vascular leak. We show here that highly purified NS1 devoid of bacterial endotoxin activity at physiologically relevant concentrations directly activated mouse macrophages and human peripheral blood mononuclear cells via toll-like receptor 4, leading to the induction and release of proinflammatory cytokines and chemokines. Thus, we identify NS1 as a dengue virus-encoded pathogen-associated molecular pattern. In an in vitro model of vascular leak we further showed that human microvascular endothelial cells responded to both LPS and NS1, with production of IL-6 and the disruption of HMEC-1 monolayer integrity. The treatment of antagonist of LPS, LPS-RS, and anti-TLR4 antibody significantly reduce these effects. The importance of TLR4 activation in vivo was confirmed by the reduction in capillary leak by a TLR4 antagonist in a mouse model of dengue virus infection. The striking similarities in cellular responses to LPS and NS1 via TLR4 suggest that NS1 is a viral counterpart of bacterial endotoxin. Similar to the role of LPS in septic shock, NS1 may contribute to vascular leak in dengue patients leading to aseptic shock.