Background <p>Acute lung injury (ALI) is a complex and life-threatening condition. In severe cases, ALI can progress to acute respiratory distress syndrome (ARDS), which is associated with high mortality. It is characterized by diffuse pulmonary parenchymal inflammation and refractory hypoxemia. Neutrophils, as pivotal immune cells in the lungs, play a critical role in the development and progression of ALI. This study aimed to determine whether S100A9 deletion alleviates LPS-induced ALI by modulating neutrophil activation through the NRF2/HO-1 signaling axis.</p> Methods <p>We established an ALI model using LPS in both wild-type (WT) and S100A9 gene knockout (KO) mice to assess pulmonary inflammation and oxidative stress. Lung injury severity, inflammation, oxidative stress status, neutrophil accumulation, and NET formation were evaluated in vivo, and bone marrow-derived primary neutrophils were exposed to LPS in vitro to examine their activation characteristics and antioxidant capacity.</p> Results <p>S100A9 KO significantly ameliorated lung tissue injury, edema, and inflammatory cell infiltration, and improved mouse survival rates. Mechanistically, S100A9 knockout alleviates acute lung injury by activating the NRF2 signaling pathway, which in turn downregulates neutrophil activation and the production of NETs.</p> Conclusion <p>Our findings indicate that S100A9 deletion attenuates neutrophil activation and oxidative stress and may alleviate LPS-induced ALI through activation of the NRF2/HO-1 signaling pathway.</p> Graphical Abstract <p></p>