Background/Objectives: Seasonal reproduction in mammals is primarily regulated by the hypothalamic–pituitary–ovarian (HPO) axis, yet its molecular mechanisms in subterranean rodents living in light-restricted environments remain poorly understood. This study aimed to characterize the transcriptional regulation of the HPO axis during seasonal estrus in the Manchurian zokor (Myospalax psilurus, M. psilurus), a fossorial rodent exhibiting distinct breeding cycles despite perpetual darkness. Methods: Hypothalamic, pituitary, and ovarian tissues were collected from female zokors during estrus and anestrus (n = 5 per group). RNA sequencing was performed, followed by de novo transcriptome assembly and bioinformatic analyses. Differentially expressed genes (DEGs) were identified using edgeR, and functional enrichment was assessed via GO and KEGG analyses. Key DEGs were validated by RT-qPCR. Results: A total of 513, 292, and 138 DEGs were identified in the hypothalamus, pituitary, and ovary, respectively. GO analysis highlighted enrichment in G-protein-coupled receptor signaling, oxidation–reduction processes, and calcium ion binding. KEGG pathway analysis revealed significant enrichment of the neuroactive ligand–receptor interaction pathway across all three tissues. Key candidate genes included Trh and Mc3r in the hypothalamus, Pitx2 and NR4A2 in the pituitary, and PTGER2 and Sphk1 in the ovary. Conclusions: This study provides the first comprehensive transcriptomic profile of the HPO axis in Manchurian zokors during seasonal estrus. The neuroactive ligand–receptor interaction pathway appears central to reproductive regulation, and several tissue-specific genes were identified as potential regulators of seasonal breeding. These findings enhance our understanding of reproductive adaptation in subterranean mammals and offer a foundation for further functional studies.