Abstract Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). In the current study we investigated the role of CD44 in ALI/ARDS. Intranasal exposure of CD44 wild type (CD44 WT) mice to SEB led to a significant increase in the expression of CD44 on lung mononuclear cells. CD44 knockout (CD44 KO) mice developed significantly reduced SEB-induced ALI/ARDS, compared to similarly treated CD44 WT mice. Deletion of CD44 did not have a significant effect on lymphocyte activation, apoptosis or inflammatory cytokine production. However, mononuclear cell migration to the lungs of SEB-exposed CD44 KO mice was significantly reduced when compared to mononuclear infiltration in the lungs of SEB-exposed CD44 WT mice. Mechanistically, deletion of CD44 led to reduced ability of SEB-exposed spleen cells to bind to lung epithelial cells. Finally, treatment of SEB-exposed mice with anti-CD44 mAbs led to a significant reduction in vascular permeability, prevented inflammatory cells infiltration in the lungs and reduced the ability of SEB-exposed splenocytes to adhere to epithelial cells. Together, these results demonstrate an important role of CD44 in SEB-induced lung injury and suggest the possibility of targeting CD44 for the treatment of SEB-induced ALI/ARDS.