Acute lung injury (ALI) is a life-threatening, diffuse heterogeneous lung injury characterized by acute onset, pulmonary edema, and respiratory failure. Lipopolysaccharide (LPS) is a leading cause for ALI and when administered to a mouse it induces a lung phenotype exhibiting some of the clinical characteristics of human ALI. This study focused on investigating whether microRNA-27b (miR-27b) affects ALI in a mouse model established by LPS-induction and to further explore the underlying mechanism. After model establishment, the mice were treated with miR-27b agomir, miR-27b antagomir, or D-ribofuranosylbenzimidazole (an inhibitor of nuclear factor-E2-related factor 2 [Nrf2]) to determine levels of miR-27b, Nrf2, nuclear factor kappa-light-chain-enhancer of activated B cells nuclear factor kappa B (NF-kappa B), p-NF-kappa B, and heme oxygenase-1 (HO-1). The levels of interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in bronchoalveolar lavage fluid (BALF) were determined. The results of luciferase activity suggested that Nrf2 was a target gene of miR-27b. It was indicated that the Nrf2 level decreased in lung tissues from ALI mice. The downregulation of miR-27b decreased the levels of IL-1 beta, IL-6, and TNF-alpha in BALF of ALI mice. Downregulated miR-27b increased Nrf2 level, thus enhancing HO-1 level along with reduction of NF-kappa B level as well as the extent of NF-kappa B phosphorylation in the lung tissues of the transfected mice. Pathological changes were ameliorated in LPS-reduced mice elicited by miR-27b inhibition. The results of this study demonstrate that downregulated miR-27b couldenhance Nrf2 and HO-1 expressions, inhibit NF-kappa B signaling pathway, which exerts a protective effect on LPS-induced ALI in mice.