The endothelial-to-mesenchymal transition (EndoMT) is a cellular process featuring decreased expression of endothelial marker genes but increased expression of mesenchymal marker genes. The EndoMT is involved in endothelial dysfunction and the pathogenesis of atherosclerosis. To investigate the dynamic expression of EndoMT genes in vascular endothelial cells under atheroprotective pulasatile shear stress (PS) and atheroprone oscillatory shear stress (OS), we analyzed RNA sequencing data from multi-timepoint shear stress experiments. This unbiased analysis involving next-generation sequencing confirmed that PS and OS had an opposite effect in regulating EndoMT genes. Further experimental validations with H2O2 and gain- and loss-of-function approaches indicated that reactive oxygen species are involved in the OS-induced EndoMT, whereas AMP-activated protein kinase and sirtuin-1 could inhibit the OS-induced EndoMT. Furthermore, as compared with PS, OS increased the DNA methylation of the promoter regions of vWF, CD31, and CDH5 genes but decreased that of CDH2, FSP1, and vimentin. The translational implication of the current study builds on the ability of the anti-diabetic drug metformin and cholesterol-lowering drug atorvastatin to suppress EndoMT in cultured endothelial cells and in mouse aortae.