Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM⁺ HDL contained S1P, whereas ApoM⁻ HDL did not. Moreover, HDL in Apom^−/− mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P–human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM⁺ HDL induced S1P₁ receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM⁻ HDL did not. Importantly, lack of S1P in the HDL fraction of Apom^−/− mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P₁ receptor on endothelial cells, is a vasculoprotective constituent of HDL.