Smad2/3 are downstream signaling molecules for TGF-β and Myostatin. Recently, Myostatin was shown to induce ROS in skeletal muscle via canonical Smad3, NF-κB and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Myostatin levels. Hence, our aims were firstly to investigate if Myostatin induced muscle atrophy in Smad3-/- mice by increasing ROS and secondly to delineate Smad3-independent signaling mechanism for Myostatin-induced ROS. Herein we show that Smad3-/- mice have increased ROS levels in skeletal muscle and inactivation of Myostatin in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Myostatin in Smad3-/-muscle was not via NF-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, Nox and XO levels were up-regulated that led to an increase in ROS production in Smad3-/- skeletal muscle. The exaggerated ROS in the Smad3-/- muscle potentiated binding of CHOP transcription factor to MuRF1 promoter resulting in enhanced MuRF1 levels leading to muscle atrophy.