The present study established a method to prepare heated tobacco-derived cigarette smoke extract (hCSE) and burned cigarette-derived CSE (bCSE), and compared their effects on human umbilical vein endothelial EA.hy926 cells. Three types of tobacco heating devices (Ploom S, glo, and IQOS) were used in order to generate cigarette smoke at different heating temperatures. hCSE and bCSE were prepared using the puffing regimen parameters of the CORESTA approach (55 mL puff volume, 3 sec puff duration, and 1 puff every 30 sec). Tar phase (nicotine and tar) of cigarette smoke was removed by passing through a Cambridge glass fiber filter. The rank order of amounts of nicotine and tar trapped on the Cambridge filter was Ploom S (6 ± 1 mg/91 puffs) < glo (16 ± 1 mg/91 puffs) < IQOS (56 ± 3 mg/91 puffs) < hi-lite (228 ± 2 mg/91 puffs). Crude hCSE and bCSE caused a decrease in mitochondrial metabolic activity and the expression level of endothelial nitric oxide synthase, with the rank order of potency as follows: Ploom S < glo < IQOS < hi-lite. The reduction in mitochondrial metabolic activity was diminished by removing nicotine and tar from cigarette smoke with Cambridge filter. These results indicated that higher cytotoxicity of cigarette smoke to vascular endothelial cells were correlated with higher heating/burning temperatures [Ploom S (200℃) < glo (240℃) < IQOS (300−350℃) < hi-lite (770−870℃)].