Background Estrogen homeostasis is crucial for the structure and function of the urethra, and estrogen deprivation resulting from menopause, ovariectomy, or ovarian dysfunction may lead to various urethral dysfunctions. However, the specific molecular mechanisms involved are still not fully understood. Methods Urethras from three ovariectomized (OVX) rats and three Sham rats were collected for snRNA-seq analysis. Data analysis included unsupervised clustering using the UMAP algorithm to identify distinct cell types based on marker gene expression. Differential gene expression analysis was performed to identify changes in estrogen-related gene expression across different cell types. Functional enrichment analysis was conducted to elucidate biological pathways associated with differentially expressed genes. Additionally, cellular interactions and developmental trajectories were analyzed to characterize cellular dynamics during menopause. Results Here, we profiled 69,529 single-nucleus transcriptomes from rat urethra (three OVX rats and three Sham rats). The snRNA-seq analysis revealed pronounced cellular heterogeneity and menopause-associated transcriptional reprogramming. We identified Fos as a key transcription factor associated with epithelial cell communication and differentiation under estrogen-deprived conditions. In addition, basal epithelial cells displayed EMT-associated transcriptional programs and a potential epithelial-to-mesenchymal continuum toward a mesenchymal-like state in OVX rats. We also identified Tmem233 as a hub gene in a striated muscle contraction-related module enriched in type IIa myofibers, and observed heightened inflammatory activation in immune cells, particularly T cells, in OVX rats. Conclusion In summary, our study provides a comparative analysis of the snRNA-seq data from the urethra of female rats, elucidating cellular and molecular changes during menopause.