Sphingosine 1-phosphate (S1P) lyase (SPL) is an intracellular enzyme that mediates the irreversible degradation of the bioactive lipid S1P. We have previously reported that overexpressed SPL displays anti-influenza viral activity; however, the underlying mechanism is incompletely understood. In this study, we demonstrate that SPL functions as a positive regulator of IKK« to propel type I IFN–mediated innate immune responses against viral infection. Exogenous SPL expression inhibited influenza A virus replication, which correlated with an increase in type I IFN production and IFN-stimulated gene accumulation upon infection. In contrast, the lack of SPL expression led to an elevated cellular susceptibility to influenza A virus infection. In support of this, SPLdeficient cells were defective in mounting an effective IFN response when stimulated by influenza viral RNAs. SPL augmented the activation status of IKK« and enhanced the kinase-induced phosphorylation of IRF3 and the synthesis of type I IFNs. However, the S1P degradation-incompetent form of SPL also enhanced IFN responses, suggesting that SPL’s pro-IFN function is independent of S1P. Biochemical analyses revealed that SPL, as well as the mutant form of SPL, interacts with IKK«. Importantly, when endogenous IKK« was downregulated using a small interfering RNA approach, SPL’s anti-influenza viral activity was markedly suppressed. This indicates that IKK« is crucial for SPL-mediated inhibition of influenza virus replication. Thus, the results illustrate the functional significance of the SPL–IKK«–IFN axis during host innate immunity against viral infection. The Journal of Immunology, 2017, 199: 000–000.