Neutrophil infiltration is an insidious feature in septic lung injury, although the specific adhesive mechanisms regulating pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. The aim of this present study was to define the role of CD44 in sepsis-induced neutrophil infiltration and lung damage. Mice were treated with a monoclonal antibody against CD44 before cecal ligation and puncture (CLP) induction. Edema formation, bronchoalveolar accumulation of neutrophils, myeloperoxidase activity, and macrophage inflammatory protein-2 (MIP-2) levels in the lung were determined after CLP. Expression of Mac-1 and CD44 on neutrophils was quantified by using flow cytometry. In separate experiments, fluorescent-labeled neutrophils co-incubated with an anti-CD44 antibody were adoptively transferred to CLP mice. CLP triggered clear-cut lung damage characterized by edema formation, neutrophil infiltration, and increased levels of MIP-2 in the lung. Notably, immunoneutralization of CD44 reduced CLP-induced pulmonary accumulation of neutrophils. In addition, functional inhibition of CD44 decreased CLP-induced lung damage and edema. However, formation of MIP-2 in the lung and neutrophil expression of Mac-1 were intact in septic mice pretreated with the anti-CD44 antibody. Adoptive transfer experiments revealed that neutrophil rather than lung CD44 mediates neutrophil accumulation in septic lung injury. Moreover, administration of hyaluronidase had no effect on CLP-induced neutrophil recruitment and tissue damage in the lung. Our data demonstrate that CD44 contributes to pulmonary infiltration of neutrophils and lung damage associated with abdominal sepsis. Thus, these novel findings suggest that CD44 may serve as a target to protect against lung injury in polymicrobial sepsis.