Myocardin is a transcriptional co-activator of serum response factor (Srf),which is a key regulator of the expression of smooth and cardiac muscle genes. Consistent with its role in regulating cardiovascular development, myocardin is the earliest known marker specific to both the cardiac and smooth muscle lineages during embryogenesis. To understand how the expression of this early transcriptional regulator is initiated and maintained, we scanned 90 kb of genomic DNA encompassing the myocardin gene for cis-regulatory elements capable of directing myocardin transcription in cardiac and smooth muscle lineages in vivo. Here, we describe an enhancer that controls cardiovascular expression of the mouse myocardin gene during mouse embryogenesis and adulthood. Activity of this enhancer in the heart and vascular system requires the combined actions of the Mef2 and Foxo transcription factors. In addition,the Tead transcription factor is required specifically for enhancer activation in neural-crest-derived smooth muscle cells and dorsal aorta. Notably,myocardin also regulates its own enhancer, but in contrast to the majority of myocardin target genes, which are dependent on Srf, myocardin acts through Mef2 to control its enhancer. These findings reveal an Srf-independent mechanism for smooth and cardiac muscle-restricted transcription and provide insight into the regulatory mechanisms responsible for establishing the smooth and cardiac muscle phenotypes during development.