Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, sphingosine, and S1P. The ubiquitous mediator S1P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca²⁺ homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK1 and SphK2 are specifically regulated are complex and only partially understood. Although SphK1 and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK1 or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.