The CRISPR/Cas9 system has been extensively used to engineer genetic loci for the generation of knockouts, insertions, and point mutations in animal models. However, many mutations that have been reported in animals are small insertions or deletions. This study used the CRISPR/Cas9 system to induce large DNA fragment deletions in MSTN via three guide RNAs in sheep. This successfully achieved the precise gene editing of the ovine MSTN gene by injecting both Cas9 mRNA and sgRNAs into embryos at the one-cell stage. Of 10 edited animals, 3 animals (30％) exhibited large genomic fragment deletions (～5 kb). Furthermore, the body weights of these 3 animals were significantly different (P0<0.0001, P15=0.001, P30=0.005, P60=0.027) between lambs with large deletions and wildtype lambs. In addition, the edited lambs were also significantly different (P0<0.0001, P15<0.0001, P30=0.002, P60=0.011) compared with wildtype. These results suggest that the generated MSTN knockout sheep is a reliable and effective animal model for further study. Furthermore, this method is time- and labor-saving, and efficient for the creation of animal models for agriculture, biology, and medicine.