Prostate cancer patients who undergo radiotherapy frequently suffer from side effects caused by radiation-induced damage to normal tissues adjacent to the tumor. Exposure of these normal cells during radiation treatment can result in tissue fibrosis and cellular senescence, which ultimately leads to postirradiation-related chronic complications including urinary urgency and frequency, erectile dysfunction, urethral stricture and incontinence. Radiation-induced reactive oxygen species (ROS) have been reported as the most potent causative factor for radiation damage to normal tissue. While MnTE-2-PyP, a ROS scavenger, protects normal cells from radiation-induced damage, it does not protect cancer cells during radiation treatment. However, the mechanism by which MnTE-2-PyP provides protection from radiation-induced fibrosis has been unclear. Our current study reveals the underlying molecular mechanism of radiation protection by MnTE-2-PyP in normal mouse prostate fibroblast cells. To investigate the role of MnTE-2-PyP in normal tissue protection after irradiation, primary prostate fibroblasts from C57BL/6 mice were cultured in the presence or absence of MnTE-2-PyP and exposed to 2 Gy of X rays. We found that MnTE-2-PyP could protect primary prostate fibroblasts from radiation-induced activation, as measured by the contraction of collagen discs, and senescence, detected by beta-galactosidase staining. We observed that MnTE-2-PyP inhibited the TGF-β-mediated fibroblast activation pathway by downregulating the expression of TGF-β receptor 2, which in turn reduced the activation and/or expression of SMAD2, SMAD3 and SMAD4. As a result, SMAD2/3-mediated transcription of profibrotic markers was reduced by MnTE-2-PyP. Due to the inhibition of the TGF-β pathway, fibroblasts treated with MnTE-2-PyP could resist radiation-induced activation and senescence. NADPH oxidase 4 (NOX4) expression is upregulated after irradiation and produces ROS. As was observed with MnTE-2-PyP treatment, NOX4-/- fibroblasts were protected from radiation-induced fibroblast activation and senescence. However, NOX4-/- fibroblasts had reduced levels of active TGF-β1, which resulted in decreased TGF-β signaling. Therefore, our data suggest that reduction of ROS levels, either by MnTE-2-PyP treatment or by eliminating NOX4 activity, significantly protects normal prostate tissues from radiation-induced tissue damage, but that these approaches work on different components of the TGF-β signaling pathway. This study proposes a crucial insight into the molecular mechanism executed by MnTE-2-PyP when utilized as a radioprotector. An understanding of how this molecule works as a radioprotector will lead to a better controlled mode of treatment for post therapy complications in prostate cancer patients.