Mammals possess reduced ability to regenerate lost tissue, compared with other vertebrates, which can regenerate through differentiation of precursor cells or de-differentiation. Mammalian multinucleated myotube formation is a differentiation process, which arises from the fusion of mononucleated myoblasts and is thought to be an irreversible process toward muscle formation. By overexpressing the Twist gene in terminally differentiated myotubes, we managed to induce reversal of cell differentiation. More specifically, following expression of the Twist gene, myotubes underwent morphological changes that caused them to cleave. This was accompanied by a reduction in the expression of certain myogenic markers. Interestingly, Twist overexpression also caused a reduction in the muscle transcription factor MyoD. Further experiments showed an increase in the cell cycle entry molecule, cyclin D1 and initiation of DNA synthesis, due to Twist overexpression. The exploitation of Twist-mediated reversal of differentiation and the study of its specific mechanism would be important in order to study mammalian cellular de-differentiation and determine its potential in muscle regeneration.