Objectives— High-density lipoprotein (HDL) levels are inversely proportional to the risk of atherosclerosis, but mechanisms of HDL atheroprotection remain unclear. Monocyte chemoatractant protein-1 (MCP-1) constitutes an early component of inflammatory response in atherosclerosis. Here we investigated the influence of HDL on MCP-1 production in vascular smooth muscle cells (VSMCs) and rat aortic explants. Methods and Results— HDL inhibited the thrombin-induced production of MCP-1 in a concentration-dependent manner. The HDL-dependent inhibition of MCP-1 production was accompanied by the suppression of reactive oxygen species (ROS), which regulate the MCP-1 production in VSMCs. HDL inhibited NAD(P)H oxidase, the preponderant source of ROS in the vasculature, and prevented the activation of Rac1, which precedes NAD(P)H-oxidase activation. The HDL capacity to inhibit MCP-1 production, ROS generation, and NAD(P)H-oxidase activation was emulated by sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), two lysosphingolipids present in HDL, but not by apolipoprotein A-I. HDL-, S1P-, and SPC-induced inhibition of MCP-1 production was attenuated in VSMCs pretreated with VPC23019, an antagonist of lysosphingolipid receptors S1P 1 and S1P 3 , but not by JTE013, an antagonist of S1P 2 . In addition, HDL, S1P, and SPC failed to inhibit MCP1 production and ROS generation in aortas from S1P 3 - and SR-B1-deficient mice. Conclusion— HDL-associated lysosphingolipids inhibit NAD(P)H oxidase-dependent ROS generation and MCP-1 production in a process that requires coordinate signaling through S1P 3 and SR-B1 receptors.