The NF-κB/iNOS pathway stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway and diverse antioxidants block muscle differentiation. Therefore, we here investigated whether Nox 2 links those two myogenic pathways in H9c2 and C 2 C 12 myoblasts. Compared with the proliferation stage, ROS generation was enhanced from the early stage of differentiation and gradually increased as differentiation progressed. Antioxidants suppressed the activated NF-κB/iNOS pathway during muscle differentiation. Nox 2 activity was also increased during muscle differentiation. Treatment with DPI and apocynin, two inhibitors of NADPH oxidase, and suppression of Nox 2 expression using siRNA, but not Nox 1, inhibited NADPH oxidase activity, muscle differentiation, and the NF-κB/iNOS pathway. Inhibition of PI 3-kinase and p38 MAPK suppressed the Nox 2/NF-κB/iNOS pathway. Nitric oxide restored muscle differentiation blocked by treatment with antioxidants or suppression of the Nox 2/NF-κB/iNOS pathway. In conclusion, Nox 2 stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway by activating the NF-κB/iNOS pathway via ROS generation.