Sphingosine 1-phosphate (S1P) can prevent endothelial cell apoptosis. We investigated the molecular mechanisms and signaling pathways by which S1P protects endothelial cells from serum deprivation-induced apoptosis. We show here that human umbilical vein endothelial cells (HUVECs) undergo apoptosis associated with increased DEVDase activity, caspase-3 activation, cytochrome c release, and DNA fragmentation after 24 h of serum deprivation. These apoptotic markers were suppressed by the addition of S1P, the NO donor S-nitroso-N-acetylpenicillamine (100 μM), or caspase-3 inhibitor z-VAD-fmk. The protective effects of S1P were reversed by the nitric-oxide synthase (NOS) inhibitor N-monomethyl-L-arginine, but not by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo[4,3-α]-quanoxaline-L-one, suggesting that NO, but not cGMP, is responsible for S1P protection from apoptosis. Furthermore, S1P increased NO production by enhancing Ca2+-sensitive NOS activity without changes in the eNOS protein level. S1P-mediated cell survival and NO production were suppressed significantly by pretreatment with antisense oligonucleotide of EDG-1 and partially by EDG-3 antisense. S1P-mediated NO production was suppressed by the addition of pertussis toxin, an inhibitor of Gi proteins, the specific inhibitor of phospholipase C (PLC), U73122, and the Ca2+ chelator BAPTA-AM. These findings indicate that S1P protects HUVECs from apoptosis through the activation of eNOS activity mainly through an EDG-1 and -3/Gi/PLC/Ca2+ signaling pathway.