AB The nuclear transcription factor hypoxia-inducible factor 1[alpha] (HIF-1α) is a key regulator of gene expression under hypoxic and inflammatory conditions. The germline-encoded pattern recognition receptor toll-like receptor 4 (TLR4) recognizes molecular motifs shared by large groups of microorganisms as well as by endogenous ligands released from stressed and/or injured tissues. We have previously demonstrated that local inhibition of HIF-1α ameliorates lung injury induced by trauma/hemorrhagic shock (T/HS) in rats. In the current study, we directly determined the role of TLR4 in HIF-1α activation during T/HS-induced acute lung injury in mice. C3H/HeJ mice that harbor a TLR4 mutation and wild-type (WT) mice were infused T/HS or trauma/sham shock (T/SS) lymph from Sprague-Dawley rats. Evans blue dye lung permeability, lung water content, myeloperoxidase levels, and lung histological analysis confirmed that TLR4-deficient mice are resistant to lung injury after T/HS lymph infusion. Lungs from WT and TLR4mut mice after T/SS lymph infusion expressed negligible levels of HIF-1α. The induction of HIF-1α in lung homogenates from TLR4mut mice after T/HS or T/HS lymph infusion was markedly reduced as compared with their WT counterparts but remained elevated as compared with TLR4mut mice after T/SS lymph infusion. Endothelial cells from TLR4mut mice and silence of TLR4 in cells from WT mice showed a remarkable reduction of HIF-1α on T/HS lymph stimulation. Blocking of nuclear factor-κB activity by SN50 or Bay 11-7085 in WT cells diminished T/HS lymph-induced HIF-1α accumulation when compared with T/SS lymph incubation. Thus, our data suggest that TLR4 activation by T/HS is necessary for T/HS-induced lung injury and an augmented pulmonary HIF-1α response, which will provide more insights into the pathogenesis of shock-induced acute lung injury and identify potential therapeutic targets.