<p>Mitochondrial impairment, accompanied by excessive reactive oxygen species (ROS) production, is a key contributor to muscle atrophy and neuromuscular disorders, leading to locomotor and respiratory failure. Antimycin A (AA), an inhibitor of the electron transport chain complex III, is an effective tool to mimic mitochondrial dysfunction, whereas 25-hydroxycholesterol (25-HC) is an immune-related oxysterol that can modulate neuromuscular activity <i>via</i> the membrane estrogen receptor α (ERα)/inositol triphosphate receptor/cytoplasmic Ca²⁺ axis. Herein, we investigated the effects of AA treatment in mouse diaphragm nerve-muscle preparations and tested the hypothesis that 25-HC can mitigate AA-induced mitochondrial damage. AA increased mitochondrial ROS production and reduced mitochondrial Ca²⁺ levels and membrane potential. This was accompanied by an elevation of extracellular H₂O₂ levels and lipid peroxidation, as well as a decline in both muscle fiber contractility and evoked exocytosis at the neuromuscular junction (NMJ). Furthermore, alterations were observed in the shape of miniature end-plate responses to the release of single neurotransmitter quanta. 25-HC, at a submicromolar concentration, inhibited AA-induced mitochondrial dysfunction and oxidative stress. Additionally, 25-HC alleviated AA-dependent functional NMJ disturbances but did not reverse the muscle fiber contraction deficit. The ability of 25-HC to decrease AA-driven mitochondrial ROS generation was blocked by a selective ERα antagonist and by chelation of cytoplasmic Ca²⁺. Thus, AA induces mitochondrial damage accompanied by oxidative stress, contractile and NMJ impairments. 25-HC can counteract AA-mediated mitochondrial dysfunction and partially restore NMJ function.</p> Graphical abstract <p></p>