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We and others have previously shown that the myocardin transcription factors play critical roles in the regulation of smooth muscle cell (SMC) differentiation marker gene expression. In a yeast 2-hybrid screen for proteins that interact with myocardin-related transcription factor-A (MRTF-A), we identified the histone 3 lysine 9 (H3K9)-specific demethylase, Jmjd1a. GST pull-down assays demonstrated that Jmjd1a bound all 3 myocardin family members, and further mapping studies showed that the jumonjiC domain of Jmjd1a was sufficient to mediate this interaction. Overexpression of Jmjd1a in multipotential 10T1/2 cells decreased global levels of di-methyl H3K9, stimulated the SM α-actin and SM22 promoters, and synergistically enhanced MRTF-A– and myocardin-dependent transactivation. Using chromatin immunoprecipitation assays, we also demonstrated that TGF-β–mediated upregulation of SMC differentiation marker gene expression in 10T1/2 cells was associated with decreased H3K9 dimethylation at the CArG-containing regions of the SMC differentiation marker gene promoters. Importantly, knockdown of Jmjd1a in 10T1/2 cells and primary rat aortic SMCs by retroviral delivery of siRNA attenuated TGF-β–induced upregulation of endogenous SM myosin heavy chain expression. These effects were concomitant with increased H3K9 dimethylation at the SMC differentiation marker gene promoters and with inhibition of MRTF-A–dependent transactivation of the SMC-specific transcription. These results suggest, for the first time, that SMC differentiation marker gene expression is regulated by H3K9 methylation and that the effects of the myocardin factors on SMC-specific transcription may involve the recruitment of Jmjd1a to the SMC-specific promoters.