Formation of new blood vessels in the adult animal (i.e., angiogenesis) is an important event for tissue repair and for tumor growth and metastasis. Angiogenesis involves the migration and proliferation of endothelial cells. We have investigated the role of the growth suppressor p27^Kip1 (p27) on endothelial cell function in vitro and angiogenesis in vivo. We have generated Ad‐TetON, a replication‐deficient adenovi‐rus that constitutively expresses the reverse tet‐respon‐sive transcriptional activator, and Ad‐TRE‐p27, which drives expression of p27 under the control of the tet response element. Western blot analysis demonstrated doxycycline‐dependent overexpression of p27 in human umbilical vein endothelial cells (HUVECs) coinfected with Ad‐TetON and Ad‐TRE‐p27, which resulted in a marked inhibition of DNA replication and cell migration in vitro. Inducible overexpression of p27 in cultured HUVECs inhibited the formation of tubelike structures and, when applied in a murine model of hind limb ischemia, reduced hind limb blood flow recovery and capillary density. These findings thus underscore a novel role of p27 in regulating endothelial cell migration in vitro and angiogenesis in vivo, suggesting a novel anti‐angiogenic therapy based on inducible p27 overexpression.