Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is up-regulated in the neointima of remodeling arteries and modulates vascular smooth muscle cell (VSMC) growth. Platelet-derived growth factor (PDGF) is the prototypic growth factor for VSMCs and plays a key role in vascular remodeling. Here, we sought to further define ESDN function in primary human VSMCs. ESDN down-regulation by RNA interference significantly enhanced PDGF-induced VSMC DNA synthesis and migration. This was associated with increased ERK1/2, Src, and PDGF receptor (PDGFR)beta phosphorylation, without altering total PDGFRbeta expression levels. In binding assays, ESDN down-regulation significantly increased (125)I-PDGF maximum binding (B(max)) to PDGF receptors on VSMCs without altering the binding constant (K(d)), raising the possibility that ESDN regulates PDGFR processing. ESDN down-regulation significantly reduced ligand-induced PDGFRbeta ubiquitination. This was associated with a significant reduction in the expression level of c-Cbl, an E3 ubiquitin ligase that ubiquitinylates PDGFRbeta. Thus, ESDN modulates PDGF signaling in VSMCs via regulation of PDGFR surface levels. The ESDN effect is mediated, at least in part, through effects on PDGFRbeta ubiquitination. ESDN may serve as a target for regulating PDGFRbeta signaling in VSMCs.