The Six1 homeoprotein belongs to the Six (sine oculis) transcription factor family, the members of which are known to act as master regulators of development. Six1 is essential for promoting myogenesis during mammalian somitogenesis. Previous studies have shown that Six1 participates in later steps of myogenic differentiation by enhancing early activation of myogenin via binding to the Mef3 site of the myogenin promoter. In the present study, however, we show that overexpression of Six1 via retroviral infection suppresses the expression of myogenin and myosin in C2C12 myoblasts, consequently retarding myogenic differentiation without affecting cell proliferation or expression of Mef2 and Mef3. These findings further demonstrate the functional role of Six1 in myogenesis.