Ionizing radiation presents an important solution for virus inactivation. However, its efficacy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivation and the underlying mechanisms remain unclear. This study demonstrates radiosensitivity and radiation-induced biological changes in SARS-CoV-2 using 20 wild-type and mutant strains. The results show that 1.2 kGy of electron beam (E-beam) or 0.9 kGy of X-ray irradiation can eliminate 99.99% of SARS-CoV-2 particles. The Delta and various Omicron variants exhibit heightened sensitivity to radiation compared to the wild-type, showing nearly 99.99 % inactivation efficiency at 1.0 and 0.8 kGy. The relationship between irradiation dose and the logarithmic reduction in virus load adheres to a dose–response model, characterized by extremely narrow windows. Spike (S) protein disruption, rather than the commonly accepted nucleic acid cleavage, is identified as the primary inactivation mechanism (triggering a conformation transition of S protein from pre-fusion to post-fusion with minimal impact on nucleic acid integrity). This study introduces the concept of targeting critical proteins in coronavirus inactivation, offering valuable insight for infectious coronavirus disease control and vaccine development.