Phenotypic switching of smooth muscle cells (SMCs) plays a central role in the development of vascular diseases such as atherosclerosis and restenosis. However, the factors regulating expression of the human myocardin (Myocd) gene, the master gene regulator of SMC differentiation, have yet to be identified. In this study, we sought to identify the critical factors regulating Myocd expression in human SMCs. Using deletion/genetic reporter analyses, an upstream repressor region (URR) was localised within the Myocd promoter, herein termed PrmM. Bioinformatic analysis revealed three evolutionary conserved Klf4 sites within the URR and disruption of those elements led to substantial increases in PrmM-directed gene expression. Furthermore, ectopic expression established that Klf4 significantly decreased Myocd mRNA levels and PrmM-directed gene expression while electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays confirmed specific binding of endogenous Klf4, and not Klf5 or Klf2, to the URR of PrmM. Platelet-derived growth factor BB (PDGF-BB), a potent inhibitor of SMC differentiation, reduced Myocd mRNA levels and PrmM-directed gene expression in SMCs. A PDGF-BB-responsive region (PRR) was also identified within PrmM, overlapping with the previously identified URR, where either siRNA knockdown of Klf4 or the combined disruption of the Klf4 elements completely abolished PDGF-BB-mediated repression of PrmM-directed gene expression in SMCs. Moreover, ChIP analysis established that PDGF-BB-induced repression of Myocd gene expression is most likely regulated by enhanced binding of Klf4 and Klf5 to a lesser extent, to the PRR of PrmM. Taken together, these data provide critical insights into the transcriptional regulation of the Myocd gene in vascular SMCs, including during SMC differentiation.