Background <p>The significance of sex differences in auditory function and vulnerability to hearing disorders has gained increasing attention. However, the underlying biological mechanisms remain unclear. Estrogen and other female hormones are known to regulate gene expression in various organs and tissues. However, their role in regulating gene expression in the cochlea remains to be determined. The current study investigated how female hormonal status, through natural fluctuations of the estrous cycle and hormonal deprivation via ovariectomy, modulates cochlear gene expression in mice.</p> Methods <p>The cochlear transcriptome was examined under two experimental conditions that alter female hormones level: the normal estrous cycle and surgical removal of the ovaries (ovariectomy), with or without subsequent estradiol treatment. RNA sequencing was used to profile cochlear transcriptomes, followed by bioinformatic analyses to identify differentially expressed genes and their associated biological processes.</p> Results <p>Our study identified a set of cochlear genes whose expression varies between the proestrus and diestrus stages of the estrous cycle. Most of these cochlear genes are autosomal protein-coding genes. There were more diestrus-biased genes compared to proestrus-biased genes. Many of these estrous cycle-regulated genes have been previously reported as hormone-responsive and encompass a range of functional categories, particularly those involved in regulating cellular function. The differentially expressed genes are primarily associated with immune-related functions. Notably, immune-related genes showed higher expression during diestrus. Our study also showed that ovariectomy altered cochlear gene expression, though it influenced only a limited number of genes. Ovariectomy-modulated genes were primarily associated with inflammatory responses and autophagy. Furthermore, several ovariectomy-induced changes were reversed by estradiol treatment, highlighting the regulatory role of this hormone in cochlear gene expression.</p> Conclusions <p>Both natural hormonal fluctuations across the proestrus and diestrus stages of the estrous cycle, as well as experimental hormone manipulation through ovariectomy and estradiol treatment, can modulate cochlear gene expression. These findings suggest that hormone-driven transcriptional changes may contribute to sex differences in auditory physiology and disease vulnerability.</p>