Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can exacerbate respiratory failure. Our previous study showed that TLR4 confers protection against hyperoxia-induced lung injury and mortality. Hsp70 has potent cytoprotective properties and has been described as a TLR4 ligand in cell lines. We sought to elucidate the relationship between TLR4 and Hsp70 in hyperoxia-induced lung injury in vitro and in vivo and to define the signaling mechanisms involved. Wild-type, TLR4(-/-), and Trif(-/-) (a TLR4 adapter protein) murine lung endothelial cells (MLECs) were exposed to hyperoxia. We found markedly elevated levels of intracellular and secreted Hsp70 from wild-type mice lungs and MLECs after hyperoxia. We confirmed that Hsp70 and TLR4 coimmunoprecipitate in lung tissue and MLECs. Hsp70-mediated NF-κB activation appears to depend upon TLR4. In the absence of TLR4, Hsp70 loses its protective effects in endothelial cells. Furthermore, these protective properties of Hsp70 are TLR4 adapter Trif dependent and MyD88 independent. Hsp70-deficient mice have increased mortality during hyperoxia, and lung-targeted adenoviral delivery of Hsp70 effectively rescues both Hsp70-deficient and wild-type mice. To our knowledge, our studies are the first to define an Hsp70-TLR4-Trif cytoprotective axis in the lung and endothelial cells. This pathway is a potential therapeutic target against a range of oxidant-induced lung injuries.