Certain unsaturated fatty acids (UFAs), cleaved from lipoproteins, are known to activate the serine/threonine protein phosphatase type 2C (PP2C) alpha- and beta-isoforms. To investigate the role of UFAs in apoptosis of endothelial cells, we cocultured human umbilical vein endothelial cells (HUVECs) with THP-1 monocytes. Phorbol-12-myristic-13-acetate (PMA)-treated THP-1 monocytes differentiated into macrophages and synthesized lipoprotein lipase (LPL), the major enzyme for hydrolysis of triglycerides. We demonstrated that LPL from THP-1 macrophages released UFAs from VLDL, which were capable of inducing apoptosis in HUVECs. Physiological concentrations of VLDL did not cause apoptosis in HUVECs, whereas the combination of VLDL with LPL-rich cell medium of THP-1 macrophages did. THP-1 macrophages and HUVECs in cocultivation did not interfere with each other. However, addition of VLDL to this coculture caused apoptosis in HUVECs. Furthermore, inhibition of LPL by adding orlistat to the culture medium and down-regulation of LPL by small interfering RNA (siRNA) reduced the extent of apoptosis of HUVECs. In conclusion, our results show that the amounts of UFAs liberated from lipoproteins are high enough to induce apoptosis in endothelial cells. This underlines the proatherogenic role of UFAs in hyperlipoproteinemias.