The formation of muscle fibers involves the sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. MyoD, myogenin, and the cyclin-dependent kinase inhibitor p21cip1 are major players in the initiation and maintenance of the differentiated state of mouse embryonic muscle cells (C2C12). The messenger RNAs encoding these three proteins contain typical AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTRs), which are known to affect the half-life of many short-lived mRNAs. HuR, an RNA-binding protein that regulates both the stability and cellular movement of ARE-containing mRNAs, interacts and stabilizes the p21cip1 message under UV stress in human RKO colorectal carcinoma cells. Here, by the use of gel shift experiments and immunoprecipitation followed by reverse transcription-PCR analysis, we show that HuR interacts with MyoD, myogenin, and p21cip1 mRNAs through specific sequences in their 3'-UTRs. To demonstrate the implication of endogenous HuR in myogenesis, we knocked down its expression in myoblasts using RNA interference and observed a significant reduction of HuR expression, associated with complete inhibition of myogenesis. Moreover, the expression of MyoD and myogenin mRNAs, as well as proteins, is significantly reduced in the HuR knockdown C2C12 cells. We were able to completely re-establish the myogenic process of these defective cells by introducing back HuR protein conjugated to a cell-permeable peptide. Finally, HuR accumulates in the cytoplasm during myogenesis. Thus, our results clearly demonstrated that endogenous HuR plays a crucial role in muscle differentiation by regulating the expression and/or the nuclear export of ARE-containing mRNAs that are essential for this process.