Prolonged mechanical ventilation (MV) results in reduced diaphragmatic maximal force production and diaphragmatic atrophy. To investigate the mechanisms responsible for MV-induced diaphragmatic atrophy, we tested the hypothesis that controlled MV results in oxidation of diaphragmatic proteins and increased diaphragmatic proteolysis due to elevated protease activity. Further, we postulated that MV would result in atrophy of all diaphragmatic muscle fiber types. Mechanically ventilated animals were anesthetized, tracheostomized, and ventilated with 21% O2 for 18 hours. MV resulted in a decrease (p < 0.05) in diaphragmatic myofibrillar protein and the cross-sectional area of all muscle fiber types (i.e., I, IIa, IId/x, and IIb). Further, MV promoted an increase (p < 0.05) in diaphragmatic protein degradation along with elevated (p < 0.05) calpain and 20S proteasome activity. Finally, MV was also associated with a rise (p < 0.05) in both protein oxidation and lipid peroxidation. These data support the hypothesis that MV is associated with atrophy of all diaphragmatic fiber types, increased diaphragmatic protease activity, and augmented diaphragmatic oxidative stress.