Acute lung injury (ALI) is a serious illness, the incidence and mortality of which are very high. Free radicals, such as hydroxyl radicals (·OH) and peroxynitrite (ONOO-), are considered to be the final causative molecules in the pathogenesis of ALI. Hydrogen, a new antioxidant, can selectively reduce ·OH and ONOO-. In the present study, we investigated the hypothesis that hydrogen inhalation could ameliorate ALI induced by intra-tracheal lipopolysaccharide (LPS, 5 mg/kg body weight). Mice were randomized into three groups: sham group (physiological saline+2% hydrogen mixed gas), control group (LPS+normal air) and experiment group (LPS+2% hydrogen mixed gas). Bronchoalveolar lavage fluid (BALF) was performed to determine the total protein concentrations and pro-inflammatory cytokines. Lung tissues were assayed for oxidative stress variables, wet/dry (W/D) ratio, histological, immunohistochemistry and Western blotting examinations. Our experiments exhibited that hydrogen improved the survival rate of mice and induced a decrease in lung W/D ratio. In addition, hydrogen decreased malonaldehyde and nitrotyrosine content, inhibited myeloperoxidase and maintained superoxide dismutase activity in lung tissues and associated with a decrease in the expression of TNF-α, IL-1β, IL-6 and total protein concentrations in the BALF. Hydrogen further attenuated histopathological alterations and mitigated lung cell apoptosis. Importantly, hydrogen inhibited the activation of P-JNK, and also reversed changes in Bax, Bcl-xl and caspase-3. In conclusion, our data demonstrated that hydrogen inhalation ameliorated LPS-induced ALI and it may be exerting its protective role by preventing the activation of ROS-JNK-caspase-3 pathway.