Forkhead transcription factors (FoxOs) play a pivotal role in controlling cellular proliferation and survival. The cellular level of these factors is tightly regulated through the phosphoinositide 3-kinase/Akt and ubiquitin-mediated degradation. However, the ubiquitin ligases responsible for the degradation of FoxO1 and the relevance of this regulation to smooth muscle cell (SMC) proliferation and survival have not been fully identified. Here we showed that overexpression of C terminus of Hsc70-interacting protein (CHIP) promoted ubiquitination and degradation of FoxO1 in SMCs in response to tumor necrosis factor-␣. Both the U-box (containing ubiquitin ligase activity) and the charged (essential for FoxO1 binding) domains within CHIP were required for CHIP-mediated FoxO1 down-regulation. Moreover, interaction and ubiquitination of FoxO1 by CHIP depended on phosphorylation of FoxO1 at Ser-256. Furthermore, overexpression of CHIP repressed FoxO1-mediated transactivation and its proapoptotic function following tumor necrosis factor-␣ treatment. In contrast, knockdown of CHIP by small interfering RNA enhanced FoxO1-mediated transactivation and its effect on SMC proliferation and survival. Taken together, our data indicate that CHIP is a negative regulator of FoxO1 activity through ubiquitin-mediated degradation, and inhibition of CHIP may serve as a potential therapeutic target for reducing proliferative arterial diseases.The atherosclerotic lesion is characterized by the endothelial dysfunction, inflammation, and accumulation of vascular smooth muscle cells (SMCs), 3 foam cells, and matrix protein and lipids in the intima (1, 2). Although the pathogenic mechanisms of atherosclerosis and restenosis are complex, the balance between proliferation and apoptosis of vascular SMCs seems to be a major factor in the progression of these diseases (1, 2). Various growth factors and cytokines are known to be involved in these processes (3-6). One important pleiotropic cytokine is tumor necrosis factor-␣ (TNF-␣), which is believed to play a key role in modulating SMC proliferation, migration, survival, or apoptosis (3-6). TNF-␣ has been shown to promote cellular proliferation and survival via phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor-B signaling pathways in several cell types (7-10). Activated Akt phosphorylates and regulates a number of downstream proapoptotic proteins, among which are the forkhead factors FoxO1, FoxO3a, and FoxO4 (formally known as FKHR, FKHRL1, and AFX, respectively) (11). The FoxO proteins are multifunctional transcription factors, which have important roles in regulating cellular differentiation, proliferation, survival in various cell lines, including cancer cells, fibroblasts, myoblasts, endothelial cells, and SMCs (3-6). Activated FoxO proteins modulate apoptosis through regulation of a number of proapoptotic proteins, inducing Bim, the TNF-related death inducing ligand, Fas ligand, and TNF-R1-associated death domain, which all are involved in apoptotic signaling (11)(12)(13)(14). Recent s...