MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-beta-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1. Using both a reporter assay and Northern blot analysis, we showed that TGF-beta1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-beta1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.