Smooth muscle cells (SMCs) retain remarkable plasticity to undergo phenotypic modulation in which the expression of smooth muscle markers is markedly attenuated while conversely expression of extracellular matrix (ECM) is dramatically up-regulated. Myocardin is perhaps the most potent transcription factor for stimulating expression of smooth muscle-specific genes; little is known, however, about whether myocardin can orchestrate ECM expression to act in concert with smooth muscle differentiation program. In this study, we demonstrated myocardin coordinate smooth muscle differentiation by inducing transcription of microRNA-143 (miR-143), which attenuates ECM versican protein expression. Previous studies have shown that versican is a chondroitin sulfate proteoglycan of the ECM that is produced by synthetic SMCs and promotes SMC migration and proliferation. Our data demonstrated that myocardin significantly represses versican expression in multiple cell lines, and this occurs through the induction of miR-143 by myocardin. By a modified reverse transcribed PCR, we found that miR-143 specifically binds to the 3'-untranslated region of versican mRNA. Reporter assays validated that miR-143 targets versican 3'-untranslated region through an evolutionarily conserved miR-143 binding site. Furthermore, overexpression of miR-143 significantly represses versican expression, whereas conversely, depletion of endogenous miR-143 results in up-regulation of versican expression. In addition, we demonstrated that myocardin represses versican through induction of miR-143. Finally, we found that the regulation of versican by miR-143 is involved in platelet-derived growth factor BB-induced SMC migration. This study provides the first evidence that myocardin, in addition to activating smooth muscle-specific genes, regulates ECM expression through induction of microRNAs during smooth muscle differentiation.