<p>Cholesterol is involved in the regulation of various signaling processes, and oxysterols are essential lipid messengers. The cholesterol derivative 25-hydroxycholesterol (25-HC) is overproduced by muscle macrophages in sarcopenia, myasthenia, and neurodegenerative diseases. Herein, we examined the effects of 25-HC on Ca²⁺ signaling and contractions of the mouse diaphragm, the main respiratory muscle. We found that 25-HC increased resting levels of cytosolic Ca²⁺ in muscle fibers. This effect was dependent on estrogen receptor α (ERα) and was mediated by Ca²⁺-efflux from intracellular stores via dantrolene-insensitive and TMB-8-sensitive channels, presumably inositol trisphosphate receptors (IP₃Rs). In addition, 25-HC suppressed diaphragm contractile responses to direct stimulation of the muscle fibers. The negative effect of 25-HC on contraction force was inhibited by blockers of ERα and Ca²⁺ mobilization. Thus, 25-HC may suppress diaphragm muscle contractility due to activation of an ERα/IP₃R/Ca²⁺_in axis in muscle fibers. At the same time, 25-HC did not significantly modify the contractions elicited by phrenic nerve stimulation and respiratory activity in vivo. We discuss that&#xa0;the previously found enhancement of neuromuscular transmission mediated by 25-HC in the diaphragm can compensate for the reduction in the muscle contractions in the case of phrenic nerve activation.</p>