Sphingolipids are a structurally unique, widespread, and diverse family of lipids. Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme required for the synthesis of all sphingolipids. Not unexpectedly, SPT is highly regulated. SPT is a multi-subunit enzyme, the level of activity of which is controlled by the regulatory subunits known as the ORMDLs. Here, we discuss how the regulation of SPT activity is accomplished by multiple mechanisms, underscoring the importance of this regulation. A rapid homeostatic regulation of SPT, monitoring cellular sphingolipid levels, is mediated by the direct binding of the central sphingolipid ceramide to the SPT/ORMDL complex. This acute regulation is overlaid by a longer-term regulation in which ORMDL is removed from the remainder of the SPT complex and trafficked for degradation, resulting in enhanced SPT activity. A third level of regulation is conferred by the inclusion of specific isoforms of the subunits of SPT into the complex. The isoform composition of the SPT complex dictates both the sensitivity of the complex to levels of cellular sphingolipid and the molecular species of sphingoid backbone that are produced. Here we discuss the mechanisms, interplay, and physiological roles of these three levels of regulation of sphingolipid biosynthesis.