KLF5 is an important regulator of cell proliferation, differentiation, and apoptosis in mammals. Little is known about the function of KLF5 in the regulation of chicken. Hence, qPCR was used to detect the expression of KLF5 in different tissues of chicken. And chicken skeletal muscle satellite cells (SMSCs) were transfected KLF5-specific small interfering RNA (siRNA) to assay SMSCs' proliferation, differentiation, and apoptosis. The results showed that KLF5 expressed higher in skeletal muscle than in the other tissues of chicken. Knockdown of KLF5 significantly inhibited the differentiation and increased apoptosis of chicken SMSCs, but it had no significant effect on proliferation of SMSCs. These results indicate that KLF5 plays an essential role during myogenesis, which will affect muscle repair and muscle regeneration, and may ameliorate muscle aging or sarcopenia.