Acute respiratory distress syndrome (ARDS) is accompanied by severe lung inflammation induced by various diseases. Despite the severity of the symptoms, therapeutic strategies have been ineffective. High mobility group box 1 (HMGB1), which was identified originally as a DNA binding protein, has been proposed as a mediator of acute lung injury. In addition to its anti-coagulant activity, recombinant thrombomodulin (rTM) possesses an ability to suppress the inflammatory response through neutralizing HMGB1. T regulatory (Treg) cells in the lungs are reported to modify innate immune responses during resolution of acute lung injury. In the present study, we investigated the therapeutic effect of rTM, and the contribution of Treg cells to this effect, in a mouse model of severe ARDS. C57BL/6 mice received sequential intratracheal administration of α-galactosylceramide (α-GalCer) and lipopolysaccharide (LPS), which resulted in the development of severe ARDS. HMGB1 levels in the lungs increased to a higher level in ARDS mice compared to those in mice treated with LPS alone. HMGB1 was expressed in the infiltrating neutrophils and macrophages in lungs. Treg cells were reduced significantly in the lungs of ARDS mice compared to those in mice treated with LPS alone. rTM administration prolonged the survival time and ameliorated the development of ARDS, which was associated with increased Treg cells and synthesis of interleukin (IL)-10 and transforming growth factor (TGF)-β in the lungs. These results suggest that HMGB1 is involved in the development of severe ARDS and rTM shows therapeutic effects through promoting the accumulation of Treg cells at the inflammatory sites.