Over-activation of autophagy due to increased levels of reactive oxygen species is a key mechanism of lipopolysaccharide-induced acute lung injury. Hydrogen-rich saline, an antioxidant, has been proved to be an effective agent for the prevention of acute lung injury, but the underlying mechanism remains unclear. Here, we investigated the mechanism through which hydrogen-rich saline prevents acute lung injury by focusing on autophagy regulation. The acute lung injury model was induced using lipopolysaccharide both in vivo and in vitro. The activation of autophagy was observed by detecting the expression of autophagy-related proteins using Western blotting. Lung histopathological changes, malondialdehyde content, wet/dry ratio, inflammatory cell count, protein content in bronchoalveolar lavage fluid, and cell viability were measured to evaluate the severity of acute lung injury. Intracellular reactive oxygen species levels were detected using dihydroethidium, which is a reactive oxygen species fluorescent probe. The results showed that the expression of Beclin-1 and microtubule-associated protein 1 light chain 3 II/I (LC3II/I ratio) was obviously increased after lipopolysaccharide administration. Pretreatment with hydrogen-rich saline markedly inhibited the expression of Beclin-1 and the LC3II/I ratio; ameliorated lung histopathological changes, malondialdehyde content and wet/dry ratio; and reduced protein content and infiltration of inflammatory cells in bronchoalveolar lavage fluid. These protective effects were also observed by pretreatment with autophagy inhibitor 3-methyladenine. Similar results were found in vitro. The production of reactive oxygen species and the activation of the AMPK/mTOR pathway were notably enhanced in MLE-12 cells after lipopolysaccharide treatment, and ameliorated by the hydrogen-rich medium. The activation of AMPK induced by lipopolysaccharide was also inhibited by pretreatment with N-acetyl-L-cysteine (a reactive oxygen species scavenger). In addition, the over-activation of autophagy was also suppressed by compound C (an AMPK inhibitor). In conclusion, hydrogen-rich saline alleviated lipopolysaccharide-induced acute lung injury by inhibiting excessive autophagy activation via the ROS/AMPK/mTOR pathway in mice. Hydrogen-rich saline may be a new therapeutic strategy for acute lung injury prevention and treatment in the future. Impact statement Acute lung injury (ALI), a common complication of many serious health issues, such as serious infection, burns, and shock, is one of the most common critical illnesses in clinical practice with a high mortality rate of 30–40%. There are still short of effective prevention and treatment measures. Evidence is growing that hydrogen-rich saline (HRS) may be an effective drug for the prevention and treatment of ALI. However, the mechanisms involved in have not been clearly understood. In this study, we investigated the underling mechanisms by focusing on autophagy regulation. The results showed that HRS ameliorated lipopolysaccharide-induced ALI in mice by inhibiting autophagy over-activation through ROS/AMPK/mTOR pathway. HRS may be a new therapeutic strategy for ALI prevention and treatment in the future.