Insulin-like growth factor binding protein (IGFBP)-5 is a conserved protein synthesized and secreted by vascular smooth muscle cells (VSMCs). IGFBP-5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival. IGFBP-5 also stimulates VSMC migration through an IGF-independent mechanism, but the molecular basis underlying this ligand-independent action is unknown. In this study, we show that endogenous IGFBP-5 or transiently expressed IGFBP-5-EGFP, but not IGFBP-4-EGFP, is localized in the nuclei of VSMCs. Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we demonstrated that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. Intriguingly, inhibition of protein secretion abolishes IGFBP-5 nuclear localization, suggesting the nuclear IGFBP-5 is derived from the secreted protein. When added exogenously, ¹²⁵I- or Cy3-labeled IGFBP-5 is capable of cellular entry and nuclear translocation. To identify potential transcriptional factor(s) that interact with IGFBP-5, a human aorta cDNA library was screened by a yeast two-hybrid screening strategy. Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP-5, no known transcription factors were found. Further motif analysis revealed that the IGFBP-5 N-domain contains a putative transactivation domain. When fused to GAL-4 DNA dinging domain and tested, the IGFBP-5 N-domain has strong transactivation activity. Mutation of the IGF binding domain or treatment of cells with IGF-I has little effect on transactivation activity. These results suggest that IGFBP-5 is localized in VSMC nucleus and possesses transcription-regulatory activity that is IGF independent. The full text of this article is available online at http://circres.ahajournals.org.