Hongxia Li, Guoqing Zhao, Ruojun Zong, Jia Liu, Liwei Huang, Juan Xiong, Xiaolong Pan, Lijuan Huang, Lili Wang, Danna Jia, Yiyan Zhao, Xiaoyuan Zhang, Pengkang Song, Le Zhao, Xuanqi Yu, Junyao Zhang, Huimin Lv, Xiaoyu Li, Hongyong Zhang, Ruigao Song
ABSTRACT Transcription Factor EB (TFEB) is widely recognized as a key transcription factor regulating lysosomal biogenesis and autophagy. Although the TFEB gene is highly expressed in the testes, the mechanism by which it affects male fertility remains unclear. Here, we report that spermatogonia‐specific deletion of TFEB in mice results in a multifaceted reproductive phenotype, including impaired fertility, compromised sperm motility, and attenuated androgen production. Immunofluorescence results showed a significant decrease in the expression levels of TNP1, a marker for spermiogenesis. Under electron microscopy, we observed abnormalities in the mitochondria of the testes and sperm. Integrated transcriptomic and biochemical analyses identified a cluster of mitochondrial‐associated genes, including Star, Slc25a48, Gss, and ROMO1, with functional enrichment pinpointing disruptions in steroidogenic flux and calcium homeostasis. In summary, our study identifies TFEB as a pivotal regulator of mitochondrial integrity in the testes, the loss of which drives male subfertility through metabolic and hormonal dysregulation.