Background: Sepsis, one of the major life-threatening conditions and responsible for approximately 40% of clinical Acute Lung Injury (ALI) patients. Nevertheless, there are no specific medications available to reduce mortality. Bilobalide, a natural bioactive component present in Ginkgo biloba, has several medicinal properties. However, the effect of Bilobalide against ALI residues unknown. And so, the therapeutic property and underlying molecular mechanism of Bilobalide was investigated against sepsis-prompted ALI in the murine model. Materials and Methods: In C57BL/6J mice model, sepsis introduced by cecal ligation and puncture (CLP) to induce lung injury. Results: Our results showed that Bilobalide treatment increased the survival rate of CLP-induced sepsis mice. Pretreatment with Bilobalide substantially inhibits the sepsis-induced lung exudation, which is exposed by measuring the wet/dry weight of lung and lung permeability of the mice. Also, Bilobalide attenuated the histopathological alterations alike alveolar hemorrhage and infiltration of inflammatory cells are assessed by hematoxylin and eosin staining, tumor necrosis factor-α, interleukin (IL)-1 β, IL-6 and macrophage inflammatory protein-2 pro-inflammatory mediators and myeloperoxidase are measured by enzyme-linked immunosorbent assay. Molecular mechanism of Bilobalide in lung inflammation whereby cyclooxygenase-2 (COX-2), induced nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), and the activation of the nuclear factor-kappa B (kB) (p65)/IkB are determined by immunoblotting technique. Moreover, pretreatment of Bilobalide significantly downregulated the expression of COX-2, iNOS, and phosphorylation of p65 and induced the IkB activation in the lung. Further, Bilobalide prevented the oxidative stress by upregulating the expression of HO-1 in lung tissues, and gene expression of sepsis-induced anti-oxidative enzymes (catalase, MnSOD, CuZnSOD, and GPx-1) in the Bilobalide-treated mice were induced dose-dependently and it determined by a quantitative-real time-polymerase chain reaction. Conclusion: Hence, we suggested Bilobalide has the ability to act as a possible therapeutic candidate against sepsis caused ALI.