In critical care patients, acute diaphragm inactivity due to mechanical ventilation (MV) initiates a process of diaphragmatic dysfunction and atrophy referred to as ventilator-induced diaphragm dysfunction (VIDD). Although mitochondrial dysfunction linked to oxidative stress plays a critical role in VIDD, the precise molecular mechanism remains poorly understood. Herein, we show, that six hours of MV resulted in a mitochondrial fission process with reduction of mitochondria size and interaction associated with increase dynamin related protein 1 (DRP1) expression; this was prevented by P110, a molecule that blocks the recruitment of DRP1 to the mitochondrial membrane. Moreover, isolated mitochondria from MV diaphragms exhibited decreased oxygen consumption and an increase of ROS production. These mitochondrial alterations were associated with a rapid oxidation of type 1 ryanodine receptor (RyR1) as well as a depletion of the stabilizing subunit calstabin 1. Subsequently, we observed that the sarcoplasmic reticulum (SR) from the MV diaphragms was leaky to Ca²⁺ and was associated with reduced diaphragmatic contractile function. All of these changes were prevented by the mitochondrial fission inhibitor molecule P110. Taken together, the results from our study show that MV induces mitochondrial fragmentation and dysfunction in the diaphragm that is associated with an up/down regulation of 320 proteins evaluated by global quantitative proteomics analysis, mainly related to mitochondrial function. These results emphasize the importance of molecules targeting mitochondrial fission/fusion balance to prevent VIDD in humans (Fig. 1).