Soluble CD14 (sCD14), a 55-kDa glycoprotein found in plasma, has been shown to act as a shuttle for bacterial LPS and phospholipids, transporting LPS and phospholipid monomers from LPS aggregates or liposomes to high density lipoprotein particles. sCD14 has also been shown to mediate the transport of LPS and phosphatidylinositol into cells. Here we show that sCD14 mediates not only the influx but also the efflux of cellular phospholipids. Addition of sCD14 enhanced efflux of cellular phospholipids labeled with [H-3]palmitic acid, [H-3]oleic acid, or [H-3]choline chloride from differentiated THP-1 monocytic cells. Efflux was dependent on the concentration of sCD14 added and was essentially complete in 30 min. The role of membrane-bound CD14 (mCD14) in lipid efflux was assessed using matched pairs of cell lines that express or fail to express this protein. While efflux was very dependent on mCD14 in U373 cells, it was not dependent on mCD14 in Chinese hamster ovary cells, suggesting a role for additional cellular proteins in determining the pathway of phospholipid efflux. A deletion mutant of sCD14 lacking the LPS binding site had less ability to efflux phospholipids than intact sCD14, suggesting that this site is needed for CD14 to serve in phospholipid transport. [H-3]Palmitate-labeled lipids released by sCD14 were precipitated with anti-CD14 then analyzed by HPLC. Phosphatidylcholine was the dominant phospholipid exported and bound to sCD14. These results demonstrate that sCD14 mediates efflux of phospholipids from cells and suggest that sCD14 contributes to phospholipid transport in blood.