Although studies have shown that 17β-estradiol (estradiol) normalized Kupffer cell function following trauma-hemorrhage, the mechanism by which E2 maintains immune function remains unclear. Activation of Toll-like receptor 4 (TLR4) initiates an inflammatory cascade, involving activation of p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and nuclear factor-κB (NF-κB). This leads to the release of proinflammatory cytokines. Thus, we hypothesized that the salutary effects of estradiol on Kupffer cell function following trauma-hemorrhage are mediated via negative regulation of TLR4-dependent p38 MAPK and NF-κB. TLR4 mutant (C3H/HeJ) and wild type (C3H/HeOuJ) mice were subjected to trauma-hemorrhage (mean BP 35 ± 5 mmHg ∼90 min, then resuscitation) or sham operation. Administration of estradiol following trauma-hemorrhage in wild type mice decreased Kupffer cell TLR4 expression as well as prevented the phosphorylation of p38 MAPK and NF-κB. This was accompanied by normalization of Kupffer cell production capacities of IL-6, TNF-α, macrophage inflammatory protein (MIP)-1α, and MIP-2 and the decrease in plasma cytokine levels. In contrast, TLR4 mutant mice did not exhibit the increase in Kupffer cell p38 MAPK and NF-κB activation, cytokine production, or the increase in circulating cytokine levels following trauma-hemorrhage. No difference was observed in activation of PI3K among groups. These results suggest that the protective effect of estradiol on Kupffer cell function is mediated via downregulation of TLR4-dependent p38 MAPK and NF-κB signaling following trauma-hemorrhage, which prevents the systemic release of cytokines. © 2006 Elsevier Ltd. All rights reserved.