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Acute lung injury (ALI) is a severe lung disease with high mortality rate. Research has highlighted that the immune response to ALI is associated with significant changes in the expression of several microRNAs (miRNAs) in the lungs. In our research, we speculated that miR-124 moderated the severity of ALI through comprehensive suppression of the mitogen-activated protein kinase (MAPK) signaling pathway activation by targeting MAPK14. A mouse model of ALI was established by array of experiments. The expression of MAPK14 and miR-124 was assessed in the tissues of ALI mice and the expression of inflammatory cytokines in ALI mice was determined. The expression of the related kinases in the MAPK signaling pathway and key cytokines in the pro-inflammatory response were assessed by a series of experiments. Immunohistochemistry and TUNEL staining were adopted to detect lung tissue cell proliferation and apoptosis in mice with ALI. MiR-124 was poorly expressed and MAPK14 was highly expressed in tissues of ALI mice. Overexpression of miR-124 or silence of MAPK14 alleviated the symptoms of ALI by down-regulating inflammatory cytokines expression, which could intrinsically suppress the expression of associated proteins in the MAPK signaling pathway and the downstream pro-inflammatory response factors, promote proliferation and inhibit apoptosis of lung tissue cells. Overexpression of MAPK14 inverted the phenotypic changes induced by overexpressing miR-124. These results indicated that miR-124 could alleviate the symptoms of ALI by inhibiting the activation of MAPK signaling pathway via subsequent targeting of MAPK14. Additionally, miR-124 may serve as a useful biomarker to alleviate the severity of septic shock-induced lung injury. • Our study reported the function of miR-124 in ALI. • miR-124 was poorly expressed and MAPK14 was highly expressed in ALI mice. • miR-124 inactivates MAPK signaling pathway by targeting MAPK14 to relieve ALI. • miR-124 promotes proliferation and inhibits apoptosis of cells by suppressing MAPK14. • Our study provides a theoretical basis for the treatment of acute lung injury. [ABSTRACT FROM AUTHOR]