Objective— The Ezrin-radixin–moesin–binding phosphoprotein 50 (EBP50) is a scaffolding protein known to regulate ion homeostasis in the kidney and intestine. Previous work showed that EBP50 expression increases after balloon injury in rat carotids. This study was designed to determine the role of EBP50 on vascular smooth muscle cells (VSMC) proliferation and the development of neointimal hyperplasia. Methods and Results— Wire injury was performed in wild type (WT) and EBP50 knockout (KO) mice. Two weeks after injury, neointima formation was 80% lower in KO than in WT mice. Proliferation of KO VSMC was significantly lower than WT cells and overexpression of EBP50 increased VSMC proliferation. Akt activity and expression of S-phase kinase protein2 decreased in KO cells resulting in the stabilization of the cyclin-dependent kinase inhibitor, p21 cip1 . Consequently, KO cells were arrested in G 0 /G 1 phase. Consistent with these observations, p21 cip1 was detected in injured femoral arteries of KO but not WT mice. No differences in apoptosis between WT and KO were observed. Conclusion— EBP50 is critical for neointima formation and induces VSMC proliferation by decreasing S-phase kinase protein2 stability, thereby accelerating the degradation of the cell cycle inhibitor p21 cip1 .