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OBJECTIVE To investigate the key role of nuclear factor E2-related factor 2 (Nrf2) in the treatment of lung injury in sepsis mice by regulating Nrf2/heme oxygenase-1 (HO-1)/high mobility group protein B1 (HMGB1) pathway. METHODS 120 male wild type (WT) and 120 Nrf2 knockout (Nrf2-KO) ICR mice were randomly divided into Sham group, H2 control group (Sham+H2 group), cecal ligation and puncture (CLP) induced sepsis model group (CLP group) and H2 intervention group (CLP+H2 group), with 30 mice in each group. The sepsis model was reproduced by CLP. The same operation was done in Sham group and Sham+H2 group except CLP. The mice in Sham+H2 group and CLP+H2 group were challenged by 2% H2 for 1 hour at 1 hour and 6 hours after operation respectively, while the mice in Sham group and CLP group only inhaled air. Twenty mice in each group were collected to observe the 7-day survival. The other mice were sacrificed at 24 hours after the reproduction of model, and the lung tissues were harvested. The activities of superoxide dismutase (SOD) and catalase (CAT) and malondialdehyde (MDA) contents were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of HO-1 and HMGB1 were determined by Western Blot, and the positive expression of HO-1 was also detected by immunofluorescence. RESULTS Compared with Sham groups, the 7-day survival rates of WT and Nrf2-KO mice in CLP groups were significantly lowered [WT: 0% (0/20) vs. 100% (20/20), Nrf2-KO: 0% (0/20) vs. 100% (0/20), both P < 0.05]; the 7-day survival rates of CLP+H2 group in WT mice were significantly higher than those of CLP group [40% (8/20) vs. 0% (0/20), P < 0.05], but there was no significant difference between CLP+H2 group and CLP group in Nrf2-KO mice [0% (0/20) vs. 0% (0/200), P > 0.05]. In WT mice, compared with Sham group, the activities of SOD and CAT in lung tissue of CLP group were decreased significantly [SOD (kU/g): 131.30±28.21 vs. 251.00±22.84, CAT (kU/g): 13.43±1.52 vs. 20.76±1.63, both P < 0.01], the MDA content, the expressions of HO-1 and HMGB1 were increased significantly [MDA (μmol/g): 6.26±1.18 vs. 4.16±0.58, HO-1/β-actin: 0.160±0.045 vs. 0.023±0.005, HMGB1/β-actin: 0.656±0.055 vs. 0.005±0.001, all P < 0.05]. Compared with CLP group, the activities of SOD, CAT and HO-1 expression in lung tissue of CLP+H2 group were significantly increased [SOD (kU/g): 220.32±35.06 vs. 131.30±28.21, CAT (kU/g): 18.95±2.49 vs. 13.43±1.52, HO-1/β-actin: 0.376±0.025 vs. 0.160±0.045, all P < 0.01], while the MDA contents and HMGB1 expressions were significantly decreased [MDA (μmol/g): 4.26±0.75 vs. 6.26±1.18, HMGB1/β-actin: 0.343±0.040 vs. 0.656±0.055, both P < 0.05]. In Nrf2-KO mice, compared with Sham group, the activity of CAT in CLP group was significantly lowered (kU/g: 12.28±1.49 vs. 19.11±1.53, P < 0.01), MDA contents and the expressions of HO-1 and HMGB1 were significantly increased [MDA (μmol/g): 6.85±0.54 vs. 4.59±0.50, HO-1/β-actin: 0.063±0.005 vs. 0.021±0.003, HMGB1/β-actin: 0.713±0.035 vs. 0.005±0.001, all P < 0.01], while there was no significant difference in SOD activity (kU/g: 114.19±9.94 vs. 135.75±28.10, P > 0.05). There was no significant difference in above parameters between CLP+H2 group and CLP group. CONCLUSIONS H2 inhibits lung injury in septic mice through Nrf2/HO-1/HMGB1 pathway. Nrf2 plays a major role in the treatment of septic lung injury by H2.