Objective: Recent studies reported that hydrogen sulfide (H2S) is an effective agent for the prevention and treatment of acute lung injury (ALI). But the underlying mechanisms have not been understood clearly. In this study, we explored the possible mechanism from the perspective of autophagy regulation. Methods: A mouse model of ALI and alveolar type II epithelial cells (MLE-12 cells) injury was induced using lipopolysaccharide (LPS). Expression of Beclin 1 and the conversion of LC3I to LC3II were detected to evaluate the activity of autophagy. Lung histopathological changes, wet/dry (W/D) ratio, pro-inflammatory cytokines TNF-alpha, 1L-1 beta and protein content in bronchoalveolar lavage fluid (BALF), cell viability and lactic dehydrogenase (LDH) in the culture medium were determined to evaluate the severity of ALI. The activity of PI3K/Akt/mTOR pathway was detected to explore the possible mechanisms involved in the regulation of autophagy by H2S. Results: The expression of Beclin 1 and the conversion of LC3I to LC3II were significantly increased after LPS treatment and reversed by H2S both in vivo and in vitro. Lung histopathological changes, W/D ratio, TNF-alpha, IL-1 beta and protein content in BALF induced by LPS were effectively ameliorated by H2S and autophagy inhibitor 3-methyladenine. The in vitro results showed that H2S and 3-methyladenine also attenuated LPS-induced cell viability decrease and LDH release. Furthermore, H2S effectively reversed LPS-induced PI3K/Akt/mTOR signaling pathway inhibition. Conclusion: Autophagy inhibition through PI3K/Akt/mTOR pathway was involved in H2S prevention of LPS-induced ALI in mice. (C) 2018 Published by Elsevier Inc.