Sirt3, one of the seven mammalian sirtuins, is a mitochondrial nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, and has been shown to control multiple key metabolic pathways. Sirt3 deacetylases and activates a large number of mitochondrial enzymes implicated in the activity of respiratory chain and ATP production, TCA and Urea cycles. We have previously shown that mitochondrial activity is importantly involved in the regulation of myoblast differentiation. Since sirt3 modulates mitochondrial activity, we have investigated its influence on myoblast differentiation. In this purpose, we have first evaluated endogen sirt3 expression during C2C12 myoblast differentiation and then we examined the effect of its overexpression or inhibition on the differentiation processes and on mitochondrial activity. We have shown that sirt3 protein expression increased after the induction of myoblast differentiation. A transient sirt3 overexpression in C2C12 myoblasts induced the expression of Myogenin and p21 even in proliferative conditions (10% of Fetal Bovine Serum). On the other hand, stable inhibition of sirt3 expression, using short hairpin sirt3 (sh sirt3) in C2C12 myoblasts resulted in: 1) abrogation of terminal differentiation reflected by a sharp decrease of the fusion index and a significant decrease of Myogenin, MyoD and Troponin T protein expression; 2) a decrease in mitochondrial density and activity reflected by alterations in PGC1-alpha, VDAC and citrate synthase expression, and a decrease in respiratory chain complexes activity and myoblast maximal respiration. These data suggest that sirt3 plays an important role in the regulation of myoblast differentiation through its influence on mitochondrial activity.