BACKGROUND AND PURPOSE The transcription factor, Kruppel-like factor 4 (KLF4), plays an important role in regulating the proliferation of vascular smooth muscle cells. This study aimed to examine the effect of rapamycin on the expression of KLF4 and the role of KLF4 in arterial neointimal formation. EXPERIMENTAL APPROACH Expression of KLF4 was monitored using real-time PCR and immunoblotting in cultured vascular smooth muscle cells. and in rat carotid arteries in vivo after balloon injury. Adenovirus-mediated overexpression and siRNA-mediated knockdown of KLF4 were used to examine the role of KLF4 in mediating the anti-proliferative role of rapamycin. KLF4-regulated genes were identified using cDNA microarray. KEY RESULTS Rapamycin induced the expression of KLF4 in vitro and in vivo. Overexpression of KLF4 inhibited cell proliferation and the activity of mammalian target of rapamycin (mTOR) and its downstream pathways, including 4EBP-1 and p70S6K in vascular smooth muscle cells and prevented the neointimal formation in the balloon-injured arteries. KLF4 up-regulated the expression of GADD45 beta, p57kip2 and p27kip1. Furthermore, knockdown of KLF4 attenuated the anti-proliferative effect of rapamycin both in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS KLF4 plays an important role in mediating the anti-proliferative effect of rapamycin in VSMCs and balloon-injured arteries. Thus, it is a potential target for the treatment of proliferative vascular disorders such as restenosis after angioplasty.