Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as alpha-smooth muscle actin (alpha SMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated alpha SMA. More importantly, S1P challenge was responsible for the functional appearance of Ca(2+) currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P(2) turned out to be critical for the pro-differentiating effect of S1P, while S1P(3) appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration.