Although studies have shown that 17-estradiol (E2) prevents neutrophil infiltration and organ damage following trauma-hemorrhage, the mechanism by which E2 inhibits neutrophil transmigration remains unknown. Macrophage migration inhibitory factor (MIF) is thought to play a central role in exacerbation of inflammation, and is associated with lung injury. MIF regulates the inflammatory response through modulation of Toll-like receptor 4 (TLR4). Activation of TLR4 results in the release of proinflammatory cytokines and chemokines, which induce neutrophil infiltration and subsequent tissue damage. We hypothesized that E2 mediates its salutary effects in lung following trauma-hemorrhage via negative regulation of MIF and modulation of TLR4 and cytokine-induced chemotaxis. C3H/HeOuJ mice were subjected to trauma-hemorrhage (mean BP 35±5 mmHg for ~ 90 min, then resuscitation) or sham operation. Mice received vehicle, E2, or E2 in combination with recombinant mouse MIF protein (rMIF). Trauma-hemorrhage increased lung MIF and TLR4 protein levels, as well as lung and systemic levels of cytokines/chemokines. Treatment of animals with E2 following trauma-hemorrhage prevented these changes. However, administration of rMIF protein with E2 abolished the E2-mediated decrease in lung TLR4 levels, lung and plasma levels of IL-6, TNF-, MCP-1, and keratinocyte-derived chemokine (KC). Administration of rMIF protein also prevented E2-mediated reduction in neutrophil influx and tissue damage in the lungs following trauma-hemorrhage. These results suggest that the protective effects of E2 on lung injury following trauma-hemorrhage are mediated via downregulation of lung MIF and TLR4-induced cytokine/chemokine production.