The muscle-specific microRNAs, miR-1 and miR-133, play important roles in muscle growth and differentiation. Here, we show that the MEF2 transcription factor, an essential regulator of muscle development, directly activates transcription of a bicistronic primary transcript encoding miR-1-2 and 133a-1 via an intragenic muscle-specific enhancer located between the miR-1-2 and 133a-1 coding regions. This MEF2-dependent enhancer is activated in the linear heart tube during mouse embryogenesis and thereafter controls transcription throughout the atrial and ventricular chambers of the heart. MEF2 together with MyoD also regulates the miR-1-2/-133a-1 intragenic enhancer in the somite myotomes and in all skeletal muscle fibers during embryogenesis and adulthood. A similar muscle-specific intragenic enhancer controls transcription of the miR-1-1/-133a-2 locus. These findings reveal a common architecture of regulatory elements associated with the miR-1/-133 genes and underscore the central role of MEF2 as a regulator of the transcriptional and posttranscriptional pathways that control cardiac and skeletal muscle development.