Smooth muscle cell (SMC) plasticity plays an important role during development and in vascular pathologies such as atherosclerosis and restenosis. It was recently shown that down-regulation of microRNA (miR)-143 and -145, which are coexpressed from a single promoter, regulates the switch from contractile to synthetic phenotype, allowing SMCs to migrate and proliferate. We show in this study that loss of miR-143/145 in vitro and in vivo results in the formation of podosomes, which are actin-rich membrane protrusions involved in the migration of several cell types, including SMCs. We further show that platelet-derived growth factor (PDGF) mediates podosome formation in SMCs through the regulation of miR-143/145 expression via a pathway involving Src and p53. Moreover, we identify key podosome regulators as targets of miR-143 (PDGF receptor alpha and protein kinase C epsilon) and miR-145 (fascin). Thus, dysregulation of the miR-143 and -145 genes is causally involved in the aberrant SMC plasticity encountered during vascular disease, in part through the up-regulation of an autoregulatory loop that promotes podosome formation.