There is no therapeutic intervention proven to prevent the acute respiratory syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes which occur during ARDS, however the mechanisms remain elusive. The platelet receptor C-type lectin-like 2 (CLEC-2) has been shown to regulate vascular integrity at sites of acute inflammation. Therefore, the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific- or hematopoietic-specific podoplanin deficient mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48h post IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2 deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.