Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 are required for oxidant induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25µM H₂O₂) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H₂O₂ significantly reduced both myotube diameter and the abundance of numerous proteins including myosin (-81%), -actinin (-40%), desmin (-79%), talin (-37%), and troponin-I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate II-spectrin following 4- and 24hrs of H₂O₂ treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10µM Leup). Utilizing siRNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3 we demonstrated that calpain-1 is required for H₂O₂ induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant induced cellular stress.