Adhesion and recruitment of blood monocytes, processes mediated by cell adhesion molecules including E-selectin, represent an early event in atherogenesis. High density lipoproteins (HDLs) were shown to inhibit cytokine-induced expression of adhesion molecules, but mechanisms underlying this effect are not fully understood. We here investigated the effects of sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), two lysosphingolipids associated with HDL, on TNF-alpha-induced E-selectin expression in human umbilical endothelial cells. We found that HDL, SPC, and LSF inhibited E-selectin expression both on mRNA and protein level. In addition, all three agents reduced the number of E-selectin molecules present on endothelial cell surface. The inhibitory effects of HDL, SPC, and LSF on TNF-alpha-induced E-selectin expression were partially reverted in the presence of suramin, an antagonist of lysosphingolipid receptor EDG-3, or pertussis toxin, an inhibitor of trimeric G proteins. In addition, inhibition of activation of protein kinase Akt with LY294002 but not inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) with U73122 abolished the restrictive effects of HDL-, SPC-, or LSF on E-selectin expression. We conclude that HDL-associated lysosphingolipids may at least partially account for the inhibitory effects of HDL on cytokine-induced expression of adhesion molecules, and that activations of G-protein-coupled receptors and protein kinase Akt are involved in this process.