Skeletal muscle development is regulated by Six1, an important myogenic transcription factor. However, the functional analysis of duck Six1 has not been reported. Here, we cloned the coding domain sequence (CDS) region of the duck Six1 gene using RT-PCR and RACE methods. Bioinformatics analysis revealed that duck Six1 CDS region comprised of 849 bp and encoded 282 amino acids and had a high degree of homology with other species, suggesting that the functions of duck Six1 gene are conserved among other animals. Real-time PCR used to determine the mRNA expression profiles of duck Six1 in different tissues and different developmental stages showed that Six1 was highly expressed in skeletal muscle and the embryonic stage. Furthermore, the eukaryotic expression vector pEGFP-duSix1 was constructed and transfected into the duck myoblasts; the MTT assay revealed an obvious increase of cell proliferation after transfection. The expression profiles of Six1, Myf5 and MyoD showed that their expression levels were significantly increased. These results together suggested that pEGFP-duSix1 vector was constructed successfully and overexpression of duck Six1 in the myoblasts could promote cell proliferation activity and significant up-regulate expression of Myf5 and MyoD.