Aim: Mechanical ventilation (MV) results in diminished diaphragm size and strength, termed ventilator-induced diaphragm dysfunction (VIDD). VID increases dependence, prolongs weaning, and increases discharge mortality rates. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway is implicated in VIDD, upregulated following MV. JAK/STAT inhibition alleviates chronic muscle wasting conditions. This study aimed to explore the therapeutic potential of Ruxolitinib, an FDA approved JAK1/2 inhibitor (JI) for the treatment of VIDD. Methods: Rats were subjected to 5 days controlled MV (CMV) with and without daily Ruxolitinib gavage. Muscle fiber size and function were assessed. RNAseq, mitochondrial morphology, respirometry, and mass spectrometry were determined. Results: CMV significantly reduced diaphragm size and specific force by 45% (p < 0.01), associated with a two-fold P-STAT3 upregulation (p < 0.001). CMV disrupted mitochondrial content and reduced the oxygen consumption rate (p < 0.01). Expression of the motor protein myosin was unaffected, however CMV alters myosin function via post-translational modifications (PTMs). Daily administration of JI increased animal survival (40% vs. 87%; p < 0.05), restricted P-STAT3 (p < 0.001), and preserved diaphragm size and specific force. JI was associated with preserved mitochondrial content and respiratory function (p < 0.01), and the reversal or augmentation of myosin deamidation PTMs of the rod and head region. Conclusion: JI preserved diaphragm function, leading to increased survival in an experimental model of VIDD. Functional enhancement was associated with maintenance of mitochondrial content and respiration and the reversal of ventilator-induced PTMs of myosin. These results demonstrate the potential of repurposing Ruxolitinib for treatment of VIDD.