Toll-like receptor 4 (TLR4) mediates mitochondrial DNA (mtDNA) damage and biogenic responses. Mitochondrial transcription factor A (Tfam) is an essential regulator for mtDNA transcription and ATP production. Increased ATP levels were associated with normalization of immune function following trauma-hemorrhage. Moreover, administration of 17β-estradiol following trauma-hemorrhage upregulates cardiac Tfam and ATP levels. We therefore hypothesized that the salutary effect of 17β-estradiol on Kupffer cell function following trauma-hemorrhage is mediated via negative regulation of TLR4, which downregulates iNOS, upregulates Tfam and mtDNA-encoded gene cytochrome c oxidase I (mtCOI), and consequently increases cellular ATP levels. Male C3H/HeN, C3H/HeOuJ (intact TLR4), and C3H/HeJ (TLR4 mutant) mice were subjected to trauma-hemorrhage (mean BP 35 ± 5 mmHg ∼90 min, then resuscitation) or sham operation. At the beginning of resuscitation, mice received 17β-estradiol (25 μg/25 g) or vehicle intravenously and were sacrificed 2 h thereafter. Kupffer cell TLR4, iNOS, IL-6 and TNF-α production capacities were increased, and ATP, Tfam, and mtCOI levels were decreased following trauma-hemorrhage. Administration of 17β-estradiol following trauma-hemorrhage prevented the increase in Kupffer cell TLR4, iNOS, and cytokine production. This was accompanied by normalized ATP, Tfam, and mtCOI levels. Furthermore, the decreased Kupffer cell ATP and mtCOI levels were not observed in TLR4 mutant mice following trauma-hemorrhage. Taken together, these findings suggest that downregulation of TLR4-dependent ATP production is critical to 17β-estradiol-mediated immunoprotection in Kupffer cells following trauma-hemorrhage. © 2007 Wiley-Liss, Inc.