Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains, but have distinct kinetic properties and sub-cellular localizations, suggesting each has a specific role(s) in sphingosine-1-phosphate (S1P) generation. Although both kinases utilize sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung-injury model in order to investigate lipopolysaccharide (LPS)-induced lung injury in SphK1 knockout (SphK1^-/-) and wild-type (WT) mice. We found that SphK1^-/- mice were much more susceptible to LPS-induced lung injury compared to their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1^-/- mice from lung injury, and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1^-/- mouse lungs further exacerbated the response to LPS, as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection, and in fact augmented the degree of LPS-induced lung injury. Microarray and real time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that in WT mice, LPS treatment caused significantly enhanced SphK1 expression (~5x) levels within 6hr, which declined back to baseline levels by 24hr post-treatment. In contrast, expression of SphK2 was gradually induced following LPS treatment, and was elevated within 24hr. Collectively, our results for the first time demonstrate distinct functional roles of the two sphingosine kinases in the regulation of LPS-induced lung injury.