High-density lipoprotein (HDL) is well known to play an essential role in the reverse cholesterol transport, in which it removes excess free cholesterol from cells and transports it to the liver where it is excreted into the bile. The reverse cholesterol transport is considered as an important anti-atherogenic function of HDL, as it prevents macrophage foam cell formation. The HDL-receptor SR-BI is known as an important mediator in this transport mechanism. Various functions of HDL can be attributed to HDL-associated sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid, which mediates many functions involved in the cardiovascular system by signaling through one of the five G protein-coupled S1P receptors (S1P1-5). The majority of studies have examined the role of S1P receptors or that of SR-BI in HDL and HDL-S1P-mediated functions. However, the potential joint involvement of both receptors in HDL-mediated biological effects has only been investigated in a limited number of studies. As an important step toward understanding a possible relationship between S1P receptors and the SR-BI-mediated removal of cholesterol from cells to acceptors, cell lines overexpressing the SR-BI receptor and/or S1P receptors were generated to study the interaction between both in the removal of cholesterol. This study is the first to identify a functional interaction of S1P1 and SR-BI receptors. S1P1 was able to downregulate SR-BI function with clear inhibitory consequences for cholesterol efflux.