Activation of the estrogen receptor-alpha (ER) mediates the vasculoprotective effects of estrogen, in part, through modulating nitric oxide (NO) production and vasodilation. Whereas inflammation is accompanied by altered vascular reactivity and underlies the pathogenesis of vascular disease, the role of the ER in the vascular responses associated with acute systemic inflammation remains poorly characterized. Contractile and relaxation responses of isolated aortic segments were investigated 12 h following intraperitoneal injection of saline or lipopolysaccharide (LPS, 10 mg/kg) in male wild-type (ER^+/+) and ER-deficient (ER^-/-) mice. As previously observed, LPS-injected ER^+/+ mice displayed reduced contractile responses to phenylephrine (PE) and enhanced vasodilation in response to acetylcholine (ACh). In contrast, aortic tissues from LPS-injected ER^-/- mice displayed enhanced contractile responses, and reduced sensitivity to ACh- and sodium nitroprusside-induced vasodilation. LPS-treatment in ER^+/+ and ER^-/- mice resulted in similar increased levels of systemic NO production and inducible NO synthase (iNOS) expression in the vascular wall. However, expression of mRNA and protein for endothelial-NOS (eNOS) and soluble guanylate cyclase (sGC and subunits) were significantly reduced in aortic tissues from LPS-treated ER^-/- animals, possibly accounting for reduced endothelial NO production and reduced smooth muscle responses to NO. These findings represent new evidence of the functional role of ER in the male vasculature and suggest that, during acute LPS-induced inflammatory responses, the ER mediates the sustained expression of the molecular machinery essential for endothelial NO synthesis (i.e. eNOS) and the vascular responses to NO (i.e. sGC).