Mechanical ventilation (MV) is a life-saving measure in many critically ill patients. However, prolonged MV results in diaphragm dysfunction that contributes to the frequent difficulty in weaning patients from the ventilator. The molecular mechanisms underlying ventilator-induced diaphragm dysfunction (VIDD) remain poorly understood. We report here that MV induces myonuclear DNA fragmentation (3-fold increase; P<0.01) and selective activation of caspase 9 (P<0.05) and Bcl2-interacting mediator of cell death (Bim; 2- to 7-fold increase; P<0.05) in human diaphragm. MV also statistically significantly down-regulates mitochondrial gene expression and induces oxidative stress. In cultured muscle cells, we show that oxidative stress activates each of the catabolic pathways thought to underlie VIDD: apoptotic (P<0.05), proteasomal (P<0.05), and autophagic (P<0.01). Further, silencing Bim expression blocks (P<0.05) oxidative stress-induced apoptosis. Overlapping the gene expression profiles of MV human diaphragm and H₂O₂-treated muscle cells, we identify Fos, FoxO1, and Stat3 as regulators of Bim expression as well as of expression of the catabolic markers atrogin and LC3. We thus identify a novel Fos/FoxO1/Stat3-Bim intrinsic apoptotic pathway and establish the centrality of oxidative stress in the development of VIDD. This information may help in the design of specific drugs to prevent this condition.