Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CEs) and triglycerides between different lipoproteins. Recent studies have shown that blocking the function of CETP can increase the level of HDL cholesterol in blood plasma and suppress the risk of cardiovascular disease. Hence, understanding the structure, dynamics, and mechanism by which CETP transfers the neutral lipids has received tremendous attention in last decade. Although the recent crystal structure has provided direct evidence of the existence of strongly bound CEs in the CETP core, very little is known about the mechanism of CE/triglyceride transfer by CETP. In this study, we explore the large scale dynamics of CETP by means of multimicrosecond molecular dynamics simulations and normal mode analysis, which provided a wealth of detailed information about the lipid transfer mechanism of CETP. Results show that the bound CEs intraconvert between bent and linear conformations in the CETP core tunnel as a consequence of the high degree of conformational flexibility of the protein. During the conformational switching, there occurred a significant reduction in hydrophobic contacts between the CEs and CETP, and a continuous tunnel traversing across the CETP long axis appeared spontaneously. Thus, our results support the recently proposed "tunnel mechanism" of CETP from cryo-EM studies for the transfer of neutral lipids between different lipoproteins. The detailed understanding obtained here could help in devising methods to prevent CETP function as a cardiovascular disease therapeutic. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.