Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding.