Background: Lung epithelial cell dysfunction induced by hyperoxia-associated oxidative stress is a prominent feature involved in the development of acute lung injury (ALI). How the underlying molecular mechanisms contributed to this process are poorly defined. In the present study, we sought to identify the role of miR-124 in hyperoxia-induced cell apoptosis and excessive inflammatory response in pulmonary epithelial cell. Methods: The miR-124 levels in pulmonary epithelial cell were assayed by qRT-PCR. MiR-124 mimics and inhibitors were transfected to gain or loss of miR-124 function. Cell proliferation was analyzed by CCK8 assay. Cell apoptosis was analyzed by flow cytometry. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. The protein levels were assayed by western blotting. Results: The results showed that miR-124 was significantly down-regulated in Beas2B cells and primary LECs upon hyperoxia exposure conditions. However, overexpression of miR-124 dramatically attenuated hyperoxia-provoked TLR4, NF-kappa B and pro-inflammatory cytokines production. In vitro, the cell viability and apoptosis was significantly reversed following transfection with miR-124 mimics in the presence of hyperoxia. Furthermore, the 3'-untranslated region (3'-UTR) of CCL2 was bound by miR-124. Conclusion: It was concluded that miR-124 inhibited hyperoxia-induced apoptosis and excessive inflammatory response in Beas2B cells and primary LECs, at least partially, through the inhibition of TLR4/NF-kappa B/CCL2 signaling cascades.