Myogenic differentiation is precisely regulated with a cascade of genes and pathways. The previous study has demonstrated the muscle-specific deletion of Nr4a1 impairs muscle growth. However, it is still unclear whether muscular Nr4a1 deletion may directly impact myoblast physiology. Here, the present study delves into the molecular mechanism of Nr4a1 in C2C12. Through the analysis of RNAseq and microarray data, Nr4a1 was identified to highly correlate with the expression of myogenic factors. In C2C12, except confirming the induction of Nr4a1 mRNA and protein levels upon the initiation of differentiation, we observed a novel shuttling phenomenon of Nr4a1 from nucleus to cytoplasm in myoblast with a higher expression of MyoD or differentiated myotubes. Furthermore, Nr4a1 over expression in C2C12 accelerates myoblasts' differentiation and increases myoblast fusion. In contrast, ablation of Nr4a1 expression in C2C12 inhibits the differentiation and fusion process. Meanwhile, in quiescent satellite cells, Nr4a1 expressed is not detected, while its protein level is highly induced in both BaCl2-induced muscle regeneration followed with satellite cells activation and satellite cells of cultured single myofiber. The mechanism may be through the Nr4a1-mediated expression of myogenic factors, e.g. MyoD and MyoG. In summary, the current investigation demonstrates that Nr4a1 is an essential myogenic factor involved in myoblast differentiation. (C) 2019 Elsevier Inc. All rights reserved.