The diaphragm is a unique skeletal muscle designed to be rhythmically active throughout life, such that its sustained inactivation by the medical intervention of mechanical ventilation (MV) represents an unanticipated physiological state in evolutionary terms. Within a short period after initiating MV, the diaphragm develops muscle atrophy, damage, and diminished strength, and many of these features appear to arise from mitochondrial dysfunction. Notably, in response to metabolic perturbations, mitochondria fuse, divide, and interact with neighboring organelles to remodel their shape and functional properties-a process collectively known as mitochondrial dynamics. Using a quantitative electron microscopy approach, here we show that diaphragm contractile inactivity induced by 6 h of MV in mice leads to fragmentation of intermyofibrillar (IMF) but not subsarcolemmal (SS) mitochondria. Furthermore, physical interactions between adjacent organellar membranes were less abundant in IMF mitochondria during MV. The profusion proteins Mfn2 and OPA1 were unchanged, whereas abundance and activation status of the profission protein Drp1 were increased in the diaphragm following MV. Overall, our results suggest that mitochondrial morphological abnormalities characterized by excessive fission-fragmentation represent early events during MV, which could potentially contribute to the rapid onset of mitochondrial dysfunction, maladaptive signaling, and associated contractile dysfunction of the diaphragm.