Abstract: The aim of this study were to obtain fibroblast cell line with Myostatin silencing, to further understand the regulation mechanism of Myostatin and to get Myostatin silencing transgenic sheep. Four siRNAs were designed according to Myostatin gene sequences and Myostatin silencing pSilencer vectors were constructed, and then transfected into fibroblasts, the silencing effects were analysised by RTPCR; A lentiviral vector with Myostatin gene silencing was constructed and then lentivirus particles was packaged. Fibroblasts were infected by lentivirus particles and positive cells were isolated by flow cytometry. Positive cells were identified by sequencing and the silencing effect was analyzed by RTPCR. An effective siRNA sequence(PSL1) was obtained, and it inhibited the Myostatin expression significantly with a rate of 90%. The virus particles which were 108 titer determined by fluorescence microscope were obtained. Positive cell line with the purity of 99% were isolated by flow cytometry. The result indicate that positive transgene cell line without resistance marker can be quickly obtained by using lentivirus and flow cytometry. This study provides a useful cell line for further research the regulation mechanism of Myostatin and get transgenic sheep.