Rapamycin is a macrolide antibiotic that inhibits vascular smooth muscle cell proliferation and migration and that is used clinically on drug-eluting stents to inhibit in-stent restenosis. Although inhibition of cell migration is an asset in preventing restenosis, it also leads to impaired stent endothelialization, a significant limitation of current drug-eluting stent technology that necessitates prolonged antiplatelet therapy. We measured the ability of rapamycin to inhibit the migration of human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAEC) toward the chemoattractant vascular endothelial cell growth factor. Although acute administration of rapamycin had no effect, exposure for 24 h inhibited HUVEC and HCAEC migration. Disruption of the mTORC2 via small interfering RNA was also effective in inhibiting HCAEC migration. Treatment of HCAECs for this period with rapamycin produced an increase in the cyclin-dependent kinase inhibitor p27(Kip), through a decrease in the targeting of the protein for degradation by phosphorylation at Thr(187). ECs isolated from a knock-in mouse expressing p27(Kip1) with a mutation of this residue to an alanine, blocking this phosphorylation, exhibited reduced migration compared with wild-type controls. Silencing of p27(Kip1) with small interfering RNA blocked the effects of rapamycin on migration and tube formation as well as RhoA activation and cytoskeletal reorganization. We conclude that prolonged exposure of ECs to rapamycin increases p27(Kip1) and in turn inhibits RhoA activation, blocking cell migration and differentiation. These data elucidate the molecular mechanism underlying regulation of p27(Kip1) protein and cell migration by rapamycin.