PURPOSE: Testis is one of the most radiosensitive tissues. Interstitial stromal cells play a supportive role in male fertility, but radiation-induced damage to those cells has not yet been well understood. We aimed to investigate radiation-induced changes in interstitial stromal cells in the testis. METHODS: Adult male C57BL/6N mice (8 weeks) received a single pelvic exposure to a relatively high dose (1 Gy) or a very high dose (8 Gy) X-ray. We collected the testicular tissues for evaluation at 1, 9, and 60 days after irradiation. RESULTS: We detected a recoverable moderate degeneration of seminiferous tubules after 1 Gy exposure but an irreversible severe damage to the testis after 8 Gy exposure. Immunostaining results revealed that 1 Gy exposure induced DNA damage at day 1, upregulated intratubular GDNF at days 1 and 9, upregulated FGF at all time points, and upregulated CSF-1R at day 9. In contrast, 8 Gy exposure induced DNA damage at days 1 and 9, upregulated intratubular GDNF at days 1 and 9, downregulated CD105 at day 60, and upregulated FGF at all time points. CONCLUSION: Radiation-induced dynamic changes to interstitial stromal cells in the testis. Upregulated interstitial CSF-1R and FGF2 may support spermatogenesis recovery after high-dose radiation.