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 κB (NF‐κB), p‐NF‐κB, and heme oxygenase‐1 (HO‐1). The levels of interleukin (IL)‐1β, IL‐6, and tumor necrosis factor‐α (TNF‐α) 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‐1β, IL‐6, and TNF‐α in BALF of ALI mice. Downregulated miR‐27b increased Nrf2 level, thus enhancing HO‐1 level along with reduction of NF‐κB level as well as the extent of NF‐κB 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‐κB signaling pathway, which exerts a protective effect on LPS‐induced ALI in mice.