HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca²⁺ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). All three elevated intracellular Ca²⁺ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S1P₃ (also known as LPB₃ and EDG3) abolished the vasodilatory effects of SPC, S1P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S1P₃-deficient mice, Akt phosphorylation and Ca²⁺ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S1P₃-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.