Background and Objectives: Myoblast differentiation involves the formation of myotubes, fusion into multinucleated syncitia, and transcription of muscle-specific genes. Various factors are required for the regulation of myogenesis. To better understand the underlying mechanisms, we examined the potential role and function of mitochondria in myoblast differentiation. Methods: The experiments were conducted using C2C12 cells induced to differentiate using serum withdrawal. Protein and mRNA expression were determined using Western blot and semi-quantitative PCR, respectively. The ratio of mitochondrial DNA to nuclear DNA (m/n DNA) was determined by comparing the expression of cytochrome c oxidase subunit I (COX I) (mitochondrial-specific) and mitochondrial transcription factor A (TFAM) (nucleus-specific). Results: C2C12 cells began to form myotubes within 3 days after serum withdrawal and demonstrated fully differentiated phenoytypes by 2 weeks. Differentiation was associated with significantly increased expression of MyoD, 慣-myosin heavy chain (MHC), and troponin T (TnT). There were also time dependent increases in m/n DNA, mitochondrial proteins, such as cytochrome C, electron transport chain complex I (ETC I), cytochrome c oxidase subunit IV (COX IV), and the expression of the mitochondria proliferator, PGC1. These data suggest a preferential increase in mitochondrial DNA and function in differentiating C2C12 myocytes. Insulin (10 nM) treatment significantly increased the mRNA expressions of MyoD, MHC, PGC1慣, COX I and cytochrome b as well as protein expressions of TnT, cytochrome c, ETC 1 and COX IV associated with increased differentiation. On the other hand, inhibiting mitochondrial respiration with myxothiazole (1 uM) significantly decreased mRNA and protein expression of above genes/proteins as well as decreasing intracellular ATP (15%, p<0.05) and myotube formation. Conclusion: These experiments indicate that myoblast differentiation is associated with increases in mitochondrial DNA and proteins, and, importantly, that alterations of mitochondrial function modulate the state of myoblast differentiation suggesting that mitochondria likely play a critical role in myogenesis.