OBJECTIVE: The urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) are a potent multifunctional system involved in vascular remodeling. The goal of the study was to unravel the mechanisms of uPA/uPAR-directed vascular smooth muscle cell (VSMC) differentiation. METHODS AND RESULTS: Using cultured human primary VSMCs, we identified a new molecular mechanism controlling phenotypic modulation in vitro and in vivo. We found that the urokinase-type plasminogen activator receptor (uPAR) acts together with the transcriptional coactivator myocardin to regulate the VSMC phenotype. uPAR, a glycosylphosphatidylinositol-anchored cell-surface receptor family member, undergoes ligand-induced internalization and nuclear transport in VSMCs. Platelet-derived growth factor receptor β and SUMOylated RanGAP1 mediate this trafficking. Nuclear uPAR associates with myocardin, which is then recruited from the promoters of serum response factor target genes and undergoes proteasomal degradation. This chain of events initiates the synthetic VSMC phenotype. Using mouse carotid artery ligation model, we show that this mechanism contributes to adverse vascular remodeling after injury in vivo. We then cultured cells on a microstructured biomaterial and found that substrate topography induced uPAR-mediated VSMC differentiation. CONCLUSIONS: These findings reveal the transcriptional activity of uPAR, controlling the differentiation of VSMCs in a vascular disease model. They also suggest a new role for uPAR as a therapeutic target and as a marker for VSMC phenotyping on prosthetic biomaterials.