Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces membrane ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of beta-adrenergic receptor kinase, indicating that the betagamma subunit of G(i) was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced membrane ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of G(i)-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of membrane ruffling and the increase in cell motility.