Myofibroblasts are one of the key cellular components involved in fibrosis of skeletal muscle as well as in other tissues. Transforming growth factor-β1 (TGF-β1) stimulates differentiation of mesenchymal cells into myofibroblasts, but little is known about the regulatory mechanisms of myofibroblastic differentiation. Since Notch2 was shown to be downregulated in TGF-β1-induced non-muscle fibrogenic tissue, we investigated whether Notch2 also has a distinctive role in myofibroblastic differentiation of myogenic cells induced by TGF-β1. TGF-β1 treatment of C2C12 myoblasts led to expression of myofibroblastic marker α-smooth muscle actin (α-SMA) and collagen I with concomitant downregulation of Notch2 expression. Overexpression of active Notch2 inhibited TGF-β1-induced expression of α-SMA and collagen I. Interestingly, transient knockdown of Notch2 by siRNA in C2C12 myoblasts and primary cultured muscle-derived progenitor cells resulted in differentiation into myofibroblastic cells expressing α-SMA and collagen I without TGF-β1 treatment. Furthermore, we found Notch3 was counter-regulated by Notch2 in C2C12 cells. These findings suggest that Notch2 is inhibiting differentiation of myoblasts into myofibroblasts with downregulation of Notch3 expression.