Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling.