Myostatin (MSTN), is a known negative regulator of myogenesis. Silencing of the function of MSTN could result in increasing muscle mass in mice. To determine the function of endogenous MSTN expression on proliferation of sheep myoblasts, a short-hairpin RNA-targeting sheep MSTN was constructed into lentiviral vector to silence endogenous MSTN expression. We demonstrated that silencing of endogenous MSTN gene with up to approximately 73.3% reduction by short hairpin RNA (shRNA) resulted in significant increase (overall 28.3%) of proliferation of primary ovine myoblasts. The upregulation of proliferation was accompanied by the decrease expression of MyoD (-37.6%, p = 0.025), myogenin (-33.1%, p = 0.049), p21 (-49.3%, p = 0.046), and Smad3 (-50.0%, p = 0.007). Silencing of myostatin using shRNA may provide a feasible approach to improve meat productivity in farm animals.