Myostatin, a member of the transforming growth factor β (TGF-β) family, is a negative regulator for muscle growth. Loss of the function of this gene is associated with the phenotype described as “double muscling”, an extreme form of muscle development characterized by a large increase in muscle mass. Two replacement vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, were constructed, linearized, and transfected into the bovine fetal fibroblasts through electroporation. 170 drug-resistant cell colonies were obtained in cell culture medium containing 600 μg/mL G418 and 50 nmol/L GCV. Targeted homologous integration occurred in colony No. 58 as identified by PCR, and the targeted colony was further confirmed by sequencing and Southern blotting. This suggested that one allele of myostatin was successfully mutagenized in bovine fetal fibroblasts.