As a highly aggressive pediatric brainstem tumor, diffuse intrinsic pontine glioma (DIPG) accounts for 10% to 20% of childhood brain tumors. The survival rate for DIPG remains very low, with a median survival time as less than one year even under radiotherapy, the current standard treatment. Moreover, over than 250 clinical trials have failed when trying to improve the survival compared to radiotherapy. The sphingolipid metabolism and related signaling pathways have been found closely related to cancer cell survival; however, the sphingolipid metabolism targeted therapies have never been investigated in DIPG. In the current study, the anti-DIPG activity of ABC294640, the only first-in-class orally available Sphingosine kinase (SphK) inhibitor was explored. Treatment with ABC294640 significantly repressed DIPG cell growth by inducing intracellular pro-apoptotic ceramides production and cell apoptosis. We also profiled ABC294640-induced changes in gene expression within DIPG cells and identified many new genes tightly controlled by sphingolipid metabolism, such as IFITM1 and KAL1. These genes are required for DIPG cell survival and display clinical relevance in DIPG patients' samples. Together, our findings in this study indicate that targeting sphingolipid metabolism may represent a promising strategy to improve DIPG treatment.