Sphingomyelin (SM), a major sphingolipid in the lipid raft microdomains of the cell membrane, is synthesized by plasma membrane-bound sphingomyelin synthase 2 (SMS2). SMS2 is required for the maintenance of plasma membrane microdomain fluidity and receptor-mediated responses to inflammation in macrophages. However, the exact mechanism of SMS2 activation in endothelial barrier disruption and lung injury is not fully understood. To define the role of SMS activation in lung injury, we hypothesized that the inhibition of SM synthesis may provide protection against acute lung injury (ALI) by preserving endothelial barrier function. Using SMS2-silencing RNA (siRNA) treatment in human pulmonary endothelial cells (HPAECs) and tricyclodecan-9-yl-xanthogenate (D609), a competitive inhibitor of SMS, and phosphatidylcholine-specific phospholipase C in a murine model of bacterial LPS injury, we studied the role of sphingomyelin synthesis in ALI. Results show that pretreating mice with D609 significantly attenuated LPS-induced lung injury, as measured by a significant decrease in wet to dry ratio, bronchoalveolar lavage fluid cell and protein counts, and myeloperoxidase activity in lung tissue. Similarly, LPS-induced endothelial barrier disruption was significantly reduced in HPAECs pretreated with D609 or SMS2 siRNA, as demonstrated by an increase in paracellular integrity on an FITC-dextran assay, by the inhibition of LPS-induced stress fibers, and by the formation of cortical actin rings and lamellipodia at the periphery. These results indicate that D609 attenuates LPS-mediated endothelial barrier dysfunction and lung injury in mice through inhibition of SMS, suggesting a novel and essential role of SMS inhibition in modulating endothelial barrier integrity via actin cytoskeletal activation, with a potential therapeutic role in ALI.