Abstract: The aim of the study was to knock out the MSTN gene in sheep, and adenoviral vector encoding zincfinger nucleases (ZFN) targeted to the exon 1 of MSTN was constructed and packaged. Based on the high transfection efficiency and nonintegration of the adenovirus combinding with the specificity and high performance of the ZFN, disruption of the MSTN in sheep was expected in the further research. DNA sequences of T2A and the two designed ZFNs monomers were amplified by PCR and inserted into the pAdTrackCMV successively. The positive ones got were named as pAdTrackZFNLT2AZFNR. Then pAdTrackZFNLT2AZFNR and pAdEasy1 were cotransfected into E. coli. BJ5183 in which homologous recombination would happen between the two plasmids, the recombinant adenoviral vector was named as pAdEasyZFNLT2AZFNR. The recombinant vector was transfected into the HEK293 subsequently to produce the final adenovirus. Finally, the adenovirus was identified by PCR and the titer was detected, then the positive ones were reduplicated. The final adenovirus were added to the culture medium of sheep fetus fibroblasts to further confirm the infectivity of the virus and identify the activity of ZFN in mammalian cells by Western blot. The adenoviral vector containing MSTNZFN expression cassette was verified by enzymatic digestion and sequencing, and ZFN could recognize and cleave the target site of MSTN. The adenoviral vector encoding ZFN could target to the MSTN in sheep, and it was constructed successfully.