The dimeric 14-3-3 protein family protects cells from apoptosis by regulating pro-apoptotic molecules. Conversely, the cationic lipid sphingosine is associated with physiological apoptosis and induces apoptosis in its own right by a largely undefined mechanism. We show here that sphingosine and 14-3-3 interact directly in the control of cell death. The binding of sphingosine to 14-3-3 proteins renders them phosphorylatable at the dimer interface, an event that abolishes the pro-survival signalling of 14-3-3. Sphingosine kinase 1 reduces availability of sphingosine for interaction with 14-3-3, thus inhibiting cell death and providing a new mechanistic insight into the role of this enzyme in cell survival and oncogenesis. Importantly, FTY720, a sphingosine analogue with apoptotic activity that is currently in phase III clinical trials for multiple sclerosis, acts in a similar manner to sphingosine in potentiating 14-3-3 phosphorylation. The biological significance of 14-3-3 phosphorylation was demonstrated with a non-phosphorylatable 14-3-3zeta mutant which retarded apoptosis induced by sphingosine and FTY720. These results demonstrate that direct association of sphingosine with 14-3-3 is required for 14-3-3 phosphorylation, and that this axis can control cell fate. Furthermore, these results suggest a new therapeutic activity for FTY720 as an anti-cancer agent based on this mechanism.