We have earlier found that the phosphatidylethanolamine derivative ,1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3 phosphatidylethanolamine (diDCP-LA-PE) promotes translocation of the glucose transporter GLUT4 towards the cell surface and facilitates glucose uptake into cells. The present study was conducted to understand the role of N-ethylmaleimide-sensitive factor (NSF) in the diDCP-LA-PE-induced GLUT4 trafficking. Like insulin, diDCP-LA-PE increased cell surface localization of GLUT4 in differentiated 3T3-L1 adipocytes, and the effect was abolished by the vesicular exocytosis inhibitorbotulinum toxin-A. diDCP-LA-PE-induced GLUT4 translocation is clearly inhibited by knocking-down NSF. These results indicate that diDCP-LA-PE stimulates vesicular GLUT4 exocytosis by interacting with NSF.