Inflammation and oxidative stress are critical pathologies that contribute to sepsis-induced acute lung injury (ALI). This study investigated the regulatory role of estrogen-related receptor alpha (ERRα) in an experimental model of sepsis-induced ALI.In vivo, a cecal ligation and puncture- (CLP-) induced ALI model was established in anesthetized rats. Animals were then randomly assigned to receive an intraperitoneal injection of vehicle or ERRαinverse agonist (XCT-790, 2.5 mg/kg). Administration of XCT-790 significantly aggravated a sepsis-induced increase in pathological damage of lung tissues, lung endothelial permeability, myeloperoxidase (MPO) activity in lung tissues, production of serum inflammatory factors, and inflammatory cell accumulation in bronchoalveolar lavage fluid. In addition, XCT-790 treatment exacerbated a CLP-induced decrease in lung superoxide dismutase and an increase in lung malondialdehyde levels.In vitro, the exposure of rat pulmonary microvascular endothelial cells (PMVECs) to lipopolysaccharide (LPS) resulted in increased endothelial permeability and reduced expression of tight junction protein ZO-1, Occludin, JAM-A, and adherens junction protein VE-cadherin, which were further deteriorated by knockdown of ERRα. In addition, LPS-triggered inflammatory factor production and increase in the expression of phosphorylated IκBαand NF-κB p65 were also exacerbated by silencing ERRαgene. Meanwhile, knockdown of ERRαdramatically promoted LPS-activated mitochondrial reactive oxygen species production and LPS-induced downregulation of Sirt3 protein levels in rat PMVECs. Taken together, our present study provides evidences that ERRαfunctions as a novel negative modulator of sepsis-induced ALI in rats. The underlying mechanisms responsible for ERRα-elicited effects are largely dependent on the regulation of inflammatory response and oxidative stress.