In this study, we identify the natural product gambo-gic acid as well as structurally related synthetic xan-thones as first-in-class covalent inhibitors of the de novo sphingolipid biosynthesis. We apply chemo-proteomics to determine that gambogic acid binds to the regulatory small subunit B of the serine palmi-toyltransferase complex (SPTSSB). We then test structurally related synthetic xanthones to identify 18 as an equally potent but more selective binder of SPTSSB and show that 18 reduces sphingolipid levels in situ and in vivo. Finally, using various biolog-ical methods, we demonstrate that 18 induces cellular responses characteristic for diminished sphingosine-1-phosphate (S1P) signaling. This study demonstrates that SPTSSB may become a viable therapeutic target in various diseases with patholog-ical S1P signaling. Furthermore, we believe that our compound will become a valuable tool for studying the sphingolipid metabolism and serve as a blueprint for the development of a new generation of sphingo-lipid biosynthesis inhibitors.