BACKGROUND: Hexafluoropropylene oxide dimer acid (GenX), a substitute for per- and polyfluoroalkyl substances, has been widely detected in various environmental matrices and foods recently, attracting great attention. However, a systematic characterization of its reproductive toxicity is still missing. This study aims to explore the male reproductive toxicity caused by GenX exposure and the potential cellular and molecular regulatory mechanisms behind it. RESULTS: Normally developing mice were exposed to GenX, and testicular tissue was subsequently analyzed and validated using single-cell RNA sequencing. Our results revealed that GenX induced severe testicular damage, disrupted the balance between undifferentiated and differentiated spermatogonial stem cells, and led to strong variation in the cellular dynamics of spermatogenesis. Furthermore, GenX exposure caused global upregulation of testicular somatic cellular inflammatory responses, increased abnormal macrophage differentiation, and attenuated fibroblast adhesion, disorganizing the somatic-germline interactions. CONCLUSIONS: In conclusion, this study revealed complex cellular dynamics and transcriptome changes in mouse testis after GenX exposure, providing a valuable resource for understanding its reproductive toxicity.