Sine oculis homeobox 1 (Six1) homeodomain transcription factor is implicated in the genesis of muscle fiber type diversity, but its regulatory mechanisms on the formation of muscle fiber type are still poorly understood. To elucidate the biological roles of Six1 gene in muscle fiber formation, we established C2C12 cell line overexpressing Six1 and determined the effects of forced Six1 expression on muscle-specific genes expression, cell proliferation, and cell cycles. Our results indicated that Six1 overexpression could significantly promote the expression of fast-type muscle genes Atp2a1, Srl, and Mylpf. Furthermore, Six1 overexpressing C2C12 cells displayed a relative lower proliferative potential, and cell cycle analysis showed that Six1 exerted its role in cell cycle primarily through the regulation of G1/S and G2/M phases. In conclusion, Six1 plays an essential role in modulation of the fast-twitch muscle fiber phenotype through up-regulating fast-type muscle genes expression, and it could suppress the proliferation of muscle cells.