Myogenic microRNAs (myomiRs) that are specifically expressed in cardiac and skeletal muscle are highly relevant to myogenic development and diseases. Discovery and elucidation of unknown myomiRs-involved regulatory pathways in muscle cells are important, but challenging due to the lack of proper molecular tools. We report here a miR-221/222-myoD-myomiRs regulatory pathway revealed by using a small-molecule probe that selectively inhibits myomiRs including miR-1, miR-133a, and miR-206. The small-molecule inhibitor screened from luciferase assay systems was found to inhibit myomiRs and differentiation of C2C12 cells. Using the small molecule as a probe, we found that the transcriptional factor myoD, which is upstream of myomiRs, was further regulated by miR-221/222. This miR-221/222-myoD-myomiRs regulatory pathway was confirmed by over-expressing or knockdown miR-221/222 in muscle cells, which respectively led to the inhibition or enhancement of myoD protein expression and subsequent downregulation or upregulation of myomiR expression.