The sphingosine kinases, SK1 and SK2, catalyse the formation of the bioactive signalling lipid, sphingosine 1-phosphate (S1P), from sphingosine. SK1 and SK2 differ in their subcellular localisation, trafficking and regulation, but the isoforms are also distinct in their selectivity toward naturally occurring and synthetic ligands as substrates and inhibitors. To date, only the structure of SK1 has been determined, and a structural basis for selectivity differences in substrate handling by SK2 has yet to be established. Here we present a structural rationale, based on homology modelling and ligand docking, to account for the capacity of SK2, but not SK1, to efficiently process the pharmacologically active substances, fingolimod (FTY720) and safingol, as substrates. We propose that two key residue differences in hSK2 (Ser305/Thr584 in place of Ala175/Ala339 in hSK1) facilitate conformational switching in the lipid head group anchor residue, Asp308 (corresponding to Asp178 in hSK1), to accommodate substrate diversity for SK2. Our analysis accounts for the contrasting behaviour of fingolimod and safingol as non-turnover inhibitors of SK1, but substrates for SK2, and the observed stereoselectivity for phosphorylation of the pro-S hydroxymethyl group of fingolimod to generate (S)-FTY720-P in vivo. We also rationalise why methylation of the pro-R hydroxymethyl of FTY720 switches the behaviour of the resulting compound, (R)-FTY720 methyl ether (ROMe), to SK2-selective inhibition. Whilst the pharmacological significance of (S)-FTY720-P is firmly established, as the active principle of fingolimod in treating relapsing-remitting multiple sclerosis, the potential importance of SK-mediated phosphorylation of other substrates, such as safingol and non-canonical naturally occuring substrates such as (4E,nZ)-sphingadienes, is less widely appreciated. Thus, the contribution of SK2-derived safingol 1-phosphate to the anti-cancer activity of safingol should be considered. Similarly, the biological role of sphingadiene 1-phosphates derived from plant-based dietary sphingadienes, which we also show here are substrates for both SK1 and SK2, merits investigation.