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Mutations in the well-known Myostatin (MSTN) produce a “double-muscle” phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of theMSTNoften produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, aMSTNDel273Cmutation withFGF5knockout sheep, in which theMSTNandFGF5dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon ofMSTN, resulting in cysteine-depleted at amino acid position 273, and theFGF5double allele mutation led to inactivation ofFGF5gene. TheMSTNDel273Cmutation withFGF5knockout sheep highlights a dominant “double-muscle” phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, theMSTNDel273Cmutation withFGF5knockout mediated the activation ofFOSL1via the MEK-ERK-FOSL1 axis. The activatedFOSL1promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the transcription of MyoD1, and resulting in smaller myotubes.