Radiation-induced brain injury (RIBI) is caused by exposure to high doses of ionizing radiation and characterized by severe cognitive dysfunction and brain necrosis. However, the pathogenesis of RIBI is not fully understood, and no effective intervention is available. This work describes a blood-brain barrier (BBB) microphysiological system (MPS), that allowed to explore the responses of BBB and distinct brain cells to radiation exposure. Following acute exposure to radiation of X-ray or γ-ray, characteristic RIBI-associated pathological responses are observed, including BBB compromise, DNA breaks, inhibited cell proliferation, cell hypertrophy, and proinflammatory cytokine release. Among the distinctive types of cells, brain endothelial cells show the highest radiosensitivity as compared to other cells in the MPS. Intriguingly, X-ray and γ-ray radiation consistently induce prominent sterile inflammation responses, especially type I interferon response, in the BBB MPS. These responses are mediated by radiation-induced mitochondrial DNA release and subsequent activation of cGAS-STING signaling pathway. Furthermore, it is found abrocitinib (JAK1 inhibitor) and idebenone (mitochondrial protectant) can attenuate radiation-induced inflammation and ameliorate injuries in the BBB MPS. These findings reveal the involvement of mitochondria-mediated sterile inflammation in RIBI pathogenesis, identifying mitochondria as a potential target for new radioprotective measures.© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.