Micro RNA (miRNA) and mitogen-activated protein kinase (MAPK) are reported as the crucial regulators of inflammatory responses in acute lung injury (ALI). This study will explore the role of the miR-342/MAPK1 axis in regulation of lipopolysaccharide (LPS)-induced ALI. We found that miR-342 was down-regulated in LPS-induced A549 cells compared with the control group with DMSO, accompanied by elevated inflammatory cytokines and apoptosis. Over-expression of miR-342 reduced LPS-induced inflammatory responses and apoptosis in LPS-stimulated A549 cells, and had a protective role in LPS-treated mice with ALI by decreasing levels of inflammatory cytokines, improving survival of mice with ALI, and ameliorating the lung permeability. Dual-luciferase reporter gene assay demonstrated that miR-342 regulated the expression of MAPK1 by directly targeting its 3' untranslated region (3'-UTR). Mechanistically, MAPK1 silencing abrogated LPS-induced inflammatory injury in A549 cells, and partially enhanced the protective effect of miR-342. Therefore, miR-342 attenuates LPS-induced ALI by targeting MAPK1 expression, thereby protecting against A549 cell injury induced by LPS and lung injury of mice with ALI.