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Acute lung injury (ALI) is a life-threatening disease caused by the severe and acute response of the lungs to a variety of direct and indirect insults. Patients with ALI are currently treated mainly with respiratory support due to inadequate understanding of ALI progression. Alveolar epithelial cells produced thymic stromal lymphopoietin (TSLP) has been proved to worsen ALI by triggering airway inflammation. However, the regulation mechanism of TSLP expression remains unclear. In this study, we identified the crucial role played by circNCLN in lipopolysaccharide (LPS)-induced ALI. We demonstrated for the first time that miR-291a-3p could directly bind to the 3'UTR of TSLP and suppress TSLP expression in alveolar epithelial cells. Mechanistically, our data identified that circNCLN acts as a molecular sponge to antagonize miR-291a-3p and thereby maintaining the expression of TSLP in alveolar epithelial cells. Importantly, targeting circNCLN by its antisense oligonucleotide (ASO) markedly alleviated LPS-induced ALI. Therefore, our results suggested that circNCLN/miR-291a-3p/TSLP axis may be an important signaling in LPS-induced ALI and circNCLN inhibition may serve as a potential treatment of ALI.