Sphingosine-1-phosphate lyase (SPL), a key enzyme of sphingolipid metabolism, catalyzes the irreversible degradation of sphingoid base phosphates. Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. There, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SPL is a pivotal enzyme regulating S1P intracellular concentrations and a promising drug target for the design of immunosuppressants. We structurally and functionally characterized yeast SPL (Dpl1p) and its first prokaryotic homolog, from Symbiobacterium thermophilum. The Dpl1p structure served as a basis for a very reliable model of Homo sapiens SPL. The above results, together with in vitro and in vivo studies of SPL mutants, reveal which residues are involved in activity and substrate binding and pave the way to studies aimed at controlling the activity of this pivotal enzyme.