3-Hydroxy 3-methylglutaryl Coenzyme A reductase (HMG-CoAR) and its end-products are crucial for insulin-induced differentiation of fetal rat myoblasts (L6) both at early and terminal stages of development. Inhibition of HMG-CoAR activity and reduction of the enzyme levels impair the expression of L6 differentiation markers and prevent myoblast fusion into multinucleated syncytia. The mechanism underlying the modulation of this crucial enzyme so that muscular differentiation can occur is poorly understood. Thus, the aim of this work was to explore the long-term regulation of HMG-CoAR in an attempt to provide a new molecular basis for the control of muscle development. All experiments were performed in L6 rat myoblasts induced to differentiate utilizing insulin. The results indicate the following: (i) at early stages of L6 differentiation, the increase in HMG-CoAR protein levels is probably due to transcriptional induction and a decrease in the enzyme degradation rate; (ii) the subsequent reduction of HMG-CoAR protein levels is related both to an increased degradation rate and reduced gene transcription, as indicated by the rise of Insig-1 levels and the subsequent decrease in the amount of n-SREBP-1; (iii) in the terminal stages of myogenesis, reduced protein levels of HMG-CoAR could be ascribed to the decrease in gene transcription while its degradation rate is not affected. By highlighting the mechanisms involved in HMG-CoAR long-term regulation during myogenesis, this work provides useful information for searching for tools to improve the regenerative ability of muscle tissue and for the development of new pharmacological treatments of myopathies. J. Cell. Biochem. 110: 392–398, 2010. © 2010 Wiley-Liss, Inc.