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Myocardin is a potent coactivator of SRF and expressed mainly in cardiomyocytes and smooth muscle cells (SMCs), Myocardin is important for SMC differentiation, but its precise role in regulating the initiation of SMC development is less clear. Constuction and function analysis of myocardin promoter luciferase reporter plasmid will provide the theory basis for researching the function of myocardin in SMC differentiation. In this study, a mouse myocardin promoter luciferase reporter construct was successfully constructed. To determine whether myocardin transcription activity were regulated by several factors, which play important roles in SMC differentiation, luciferase repoeter assays were performed in COS-7 cells and vascular smooth muscle cells (VSMCs). The results illustrated that Smad2 significantly activated myocardin promoter, but Smad3 signaling was inhibitory to myocardin expression at 18h. Myocardin promoter transactivity was inhibited by estrogen receptor α (ERα), and enhanced by p300. These findings indicated myocardin might be regulated by multi-signals and these factors might affect SMC differentiation through activating or inhibiting myocardin transcription.