Myocardin is a transcriptional co-activator of serum response factor (SRF) and can be degraded through ubiquitin–proteasome system. Our preliminary studies unexpectedly revealed that accumulation of myocardin in response to proteasome inhibition by MG132 or lactacystin resulted in decrease of transcriptional activity of myocardin as indicated by reduced expression of SMC contractile marker genes (SM α-actin, SM22, and calponin) and muscle-enriched microRNAs (miR-143/145 and miR-1/133a), and reduced contractility of human vascular smooth muscle cells (SMCs) embedded in collagen gel lattices, suggesting that myocardin degradation is required for its transcriptional activity. Further studies using chromatin immunoprecipitation assay revealed that proteasome inhibition, although increased the occupancy of myocardin and SRF on the promoter of SM α-actin gene, abolished myocardin-dependent recruitment of RNA polymerase II. We further examined the degradation of myocardin in epithelioid and spindle-shaped SMCs and revealed that myocardin in more differentiated spindle-shaped SMCs was more quickly degraded and had shorter half-life than in epithelioid SMCs. In neointimal lesions, we found that stabilization of myocardin protein was companied by downregulation of transcripts of ubiquitin and proteasome subunits, further illustrating the mechanism underlying reduction of myocardin transcriptional activity. In summary, our results have suggested that proteasomal degradation of myocardin is required for its transcriptional activity. J. Cell. Physiol. 226: 1897–1906, 2011. © 2010 Wiley-Liss, Inc.