Myofibroblasts are one of the key cellular components involved in fibrosis of skeletal muscle as well as in other tissues. Transforming growth factor-beta1 (TGF-beta1) 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-beta1-induced non-muscle fibrogenic tissue, we investigated whether Notch2 also has a distinctive role in myofibroblastic differentiation of myogenic cells induced by TGF-beta1. TGF-beta1 treatment of C2C12 myoblasts led to expression of myofibroblastic marker alpha-smooth muscle actin (alpha-SMA) and collagen I with concomitant downregulation of Notch2 expression. Overexpression of active Notch2 inhibited TGF-beta1-induced expression of alpha-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 alpha-SMA and collagen I without TGF-beta1 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.