Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) played a prominent role in regulating muscle fiber type switching and determination. However, the role of PGC-1α in chicken muscle has seldom been explored. To investigate the effect of PGC-1α on chicken skeletal muscles in this study, the PGC-1α gene was overexpressed or silenced in chicken primary myoblasts by using lentivirus, and then the effects of the PGC-1α gene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation. The results showed that overexpression of PGC-1α in cultures in the process from proliferation to differentiation was accompanied by the upregulated expression of Pax7, MyoD, and CnAα, which was significantly (P0.05) on the expression of CnB1, NFATc3, and MyHC FRM during myofiber formation. The effective silence (P0.05) on the expression of CnB1, NFATc3, and MyHC FRM. These results indicated that the role of PGC-1α in regulating the fiber specificity of chicken skeletal muscles might be similar to that in mammals, which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.