Human umbilical cord stromal mesenchymal stem cells (hUCS-MSCs) have the potential to differentiate into numerous cell types including epithelial cells, neurons and hepatocytes in vitro, in addition to mesenchyme-derived cells such as osteocytes, chondrocytes and adipocytes. One important property of these cells is the lack of type II major histocompatibility complex class molecules, thus allowing them to be considered as an excellent candidate for transplantations. Besides the use of 5-azacytidine as a supraphysiological inducer of myogenic transformation, no study has been published to date addressing the myogenic transformation efficiency of hUCS-MSCs by using a gene transfection strategy and/or co-culture with muscle cell lines. Here, we demonstrate the reprogramming efficiency of these cells, which differentiate into myocytes in vitro by MyoD transcription factor, the master regulator of skeletal muscle differentiation. Once induced via MyoD expression, hUCS-MSCs exhibited many cellular signs of myogenic conversion within 5 days and became capable of forming multinucleated myofibers, which exhibited all functional markers of fusion machinery such as beta-catenin, neural cell adhesion molecule and M-cadherin as well as muscle cell-specific structural proteins including desmin, alpha-actinin, dystrophin, myosin heavy chain, and myoglobin together with muscle-specific enzyme, creatinine phosphokinase. Furthermore, programmed hUCS-MSCs were also capable of fusing with rat primary myoblasts to form heterokaryonic myotubes. Taken together, this study demonstrates the success of a novel cell reprogramming approach to be further evaluated at the in vivo level for use in restoring the defective dystrophin function as intrinsically found in the skeletal muscle fibers of Duchenne muscular dystrophy patients.