Myostatin (MSTN), a transforming growth factor (TGF)-β superfamily member, has been shown to negatively regulate muscle growth by inhibiting muscle precursor cell proliferation. Here, we stably transfected C2C12 cells with mouse MSTN cDNA to investigate its possible role in myoblast differentiation. We found that MSTN cDNA overexpression reversibly inhibits the myogenic process by downregulating mRNA levels of the muscle regulatory factors myoD and myogenin, as well as the activity of their downstream target creatine kinase. Taking into consideration that MSTN expression during development is restricted to muscle, our results suggest that MSTN probably regulates myogenic differentiation by an autocrine mechanism.