Neointima hyperplasia is one of the predominant features of cardiovascular diseases such as atherosclerosis, and is also responsible for the restenosis of vascular surgery including arteriovenous fistula and stent implantation. Endothelial-to-mesenchymal transition (EndMT) contributes to neointima hyperplasia by activation of the Notch or TGF-β signaling pathway. Rapamycin has been utilized as anti-restenosis drug due to its anti-proliferative activity. However, its effects on the EndMT have not been investigated yet. Thus, we examined the biological effects of rapamycin on the EndMT and its potential mechanisms. We showed that rapamycin significantly reversed TGF-β1 stimulated EndMT by upregulating endothelial marker CD31 expression and downregulating mesenchymal marker SMA-α expression in human umbilical vein endothelial cells (HUVECs). Rapamycin also inhibited TGF-β1 induced expression of the Notch signaling pathway components expression, such as Notch-1, Jagged-1, RBP-jκ and Hes-5. Among the different Notch receptors and ligands, Jagged-1/Notch-1 cascade was most remarkably blocked by rapamycin. Finally, consistently with the results from Notch inhibitor DAPT treatment, rapamycin suppressed the migration of HUVECs in vitro. Together, these findings indicate that rapamycin may function as an effective inhibitor of the EndMT in HUVECs by suppressing targeting the Notch signaling pathway.