MicroRNA (miR)-9-5 p has been shown to affect lung cancer progression and lung fibrosis, but the efficacy of miR-9-5 p in acute lung injury (ALI) remained indefinite. The study was performed to probe the modulating mechanism of miR-9-5 p in ALI via regulating macrophage polarization. The ALI mouse model was established and blood samples of ALI patients were obtained. MiR-9-5 p levels in ALI mice and ALI patients were detected. Mouse pulmonary macrophages were extracted from bronchoalveolar lavage fluid and polarized into M1 and M2 macrophages. Intervention of miR-9-5 p expression was performed to observe the effects on M1 polarization and M2 polarization in lung macrophages, inflammatory factors in BALF, wet/dry weight ratio (W/D) in lung tissues, myeloperoxidase (MPO) activity in lung tissues, and lung tissue lesion condition. MiR-9-5 p levels were elevated in the lung tissues of ALI mice and ALI patients. MiR-9-5 p silencing could repress lung macrophages in ALI mice polarized toward the M1 phenotype and promoted the polarization toward the M2 phenotype, reduced the lung lesions, the lung water content, and the secretion levels of the pro-inflammatory factors TNF-α, IL-6, and IL-1β in BALF, increased the secretion of the anti-inflammatory factor IL-10, as well as impeded the MPO activity in the lung tissues of ALI mice. MiR-9-5 p deletion ameliorates LPS-induced inflammatory infiltration in lung tissues via inhibiting the polarization of mouse lung macrophages to the M1 phenotype and promoting the polarization to the M2 phenotype.