Hydrogen gas (H2) has antioxidative, anti-inflammatory, and antiapoptotic effects and may have beneficial effects in severe sepsis. The purpose of this study was to investigate the mechanisms underlying these protective effects. Male Institute for Cancer Research mice were randomized into 6 groups: sham; sham + H2; severe sepsis; severe sepsis + H2; severe sepsis + zinc protoporphyrin IX (ZnPPIX), a heme oxygenase-1 (HO-1) inhibitor; and severe sepsis + H2 + ZnPPIX. Cecal ligation and puncture (CLP) was used to induce sepsis. Mice in the H2 groups received inhaled 2% H2 for 1 h at 1 h and 6 h after CLP or sham operation. Mice in the ZnPPIX groups received 40-mg/kg ZnPPIX by intraperitoneal injection 1 h before CLP. Tin protoporphyrin IX (TinPPIX), another HO-1 inhibitor, was also used in part for this study. Mice in the TinPPIX groups received 50-mg/kg TinPPIX through subcutaneous injection 6 h before CLP. The levels of biochemical markers, oxidative products, inflammatory mediator, the number of intestinal apoptotic cells, and the colony-forming unit numbers in the peritoneal lavage fluid were much higher in the severe sepsis group compared with the sham group. Intestinal injury in animals with severe sepsis was worse than that in animals in the sham group. H2 therapy in the animals with severe sepsis was associated with reduced intestinal injury, decreased numbers of colony-forming unit and apoptotic cells, reduced levels of biochemical markers, oxidative products, and high-mobility group box 1 protein. The protective effects of H2 were reversed by ZnPPIX and TinPPIX. Protein and messenger RNA expressions of HO-1 and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) in the intestine were increased in the severe sepsis group compared to the sham group, and H2 further increased their expressions in the severe septic mice. Zinc protoporphyrin IX and TinPPIX inhibited the expression of HO-1 protein. Hydrogen has the capacity to protect mice from organ injury in severe sepsis through a mechanism involving HO-1.