We have previously shown that several components of the RhoA signaling pathway control smooth muscle cell (SMC) phenotype by altering serum response factor (SRF)-dependent gene expression. Because our genome-wide analyses of chromatin structure and transcription factor binding suggested that the actin depolymerizing factor, destrin (DSTN), was regulated in a SMC-selective fashion, the goals of the current study were to identify the transcription mechanisms that control DSTN expression in SMC and to test whether it regulates SMC function. Immunohistochemical analyses revealed strong and at least partially SMC-selective expression of DSTN in many mouse tissues, a result consistent with human data from the genotype-tissue expression (GTEx) consortium. We identified several regulatory regions that control DSTN expression including a SMC-selective enhancer that was activated by myocardin-related transcription factor-A (MRTF-A), recombination signal binding protein for immunoglobulin κ-J region (RBPJ), and the SMAD transcription factors. Indeed, enhancer activity and endogenous DSTN expression were upregulated by RhoA and transforming growth factor-ß (TGF-ß) signaling and downregulated by inhibition of Notch cleavage. We also showed that DSTN expression was decreased in vivo by carotid artery injury and in cultured SMC cells by platelet-derived growth factor-BB (PDGF-BB) treatment. siRNA-mediated depletion of DSTN significantly enhanced MRTF-A nuclear localization and SMC differentiation marker gene expression, decreased SMC migration in scratch wound assays, and decreased SMC proliferation, as measured by cell number and cyclin-E expression. Taken together our data indicate that DSTN is a negative feedback inhibitor of RhoA/SRF-dependent gene expression in SMC that coordinately promotes SMC phenotypic modulation. Interventions that target DSTN expression or activity could serve as potential therapies for atherosclerosis and restenosis.NEW & NOTEWORTHY First, DSTN is selectively expressed in SMC in RhoA/SRF-dependent manner. Second, a SMC-selective enhancer just upstream of DSTN TSS harbors functional SRF, SMAD, and Notch/RBPJ binding elements. Third, DSTN depletion increased SRF-dependent SMC marker gene expression while inhibiting SMC migration and proliferation. Taken together, our data suggest that DSTN is a critical negative feedback inhibitor of SMC differentiation.