We identified S100A4 as a marker of rhomboid (R) smooth muscle cells (SMCs) in vitro (the synthetic phenotype, typical of intimal SMCs) in the porcine coronary artery and of intimal SMCs in vivo in both pigs and humans. S100A4 is an intracellular Ca(2+) signaling protein and can be secreted; it has extracellular functions via the receptor for advanced glycation end products (RAGE). Our objective was to explore the role of S100A4 in SMC phenotypic change, a phenomenon characteristic of atherosclerotic plaque formation. Transfection of a human S100A4-containing plasmid in spindle-shaped (S) SMCs (devoid of S100A4) led to approximately 10% of S100A4-overexpressing SMCs, S100A4 release, and a transition towards a R-phenotype of the whole SMC population. Furthermore treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a R-phenotype, which was associated with decreased SMC differentiation markers and increased proliferation and migration by activating the urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). It yielded NF-κB activation in a RAGE-dependent manner. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulation of SMC differentiation markers. By contrast, silencing of S100A4 mRNA in R-SMCs did not change the level of extracellular S100A4 or SMC morphology in spite of decreased proliferative activity. Our results show that extracellular S100A4 plays a pivotal role in SMC phenotypic changes. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.