RNA-binding Fox protein 1 (Rbfox1) controls gene expression at various levels: as a transcription co-factor, a splicing factor, and a regulator of mRNA stability and translation. Rbfox1 and its vertebrate orthologues have critical functions during development.Rbfox1mRNA, during adult myogenesis inDrosophila,exhibits bimodal expression, suggestive of the presence of a negative feedback loop. However, there are no known negative regulators ofRbfox1expression for this context. In this study, we show that the microRNAmir-9afunctions as a repressor ofRbfox1expression. Furthermore, the expression ofmir-9ais regulated by the expression of the identity transcription factor Nau. Nau and its targetmir-9aare expressed at mid myogenesis stage, and serve to restrict the expression ofRbfox1, and its targetsStat92Eandzfh1, to the early and the late myogenesis stages, as their ectopic expression is detrimental to early myofibrillogenesis. Zfh1, in turn, repressesmir-9aexpression, completing the feedback loop. Therefore, our findings identify the mechanism by which the temporal expression of the developmental geneRbfox1and its targets are tightly regulated, essential for their functions during myogenesis.Summary statementUsingDrosophila, we show that a microRNA-mediated negative feedback loop controls the expression of genes critical for skeletal muscle development.