<p>Mitochondrial dysfunction of mesenchymal stem cells (MSCs) has been implicated in impaired osteogenesis, resulting in bone loss following radiation therapy. However, the underlying mechanisms remain to be fully elucidated. This study reveals the critical role of Fis1 in regulating mitochondrial dynamics and MSC osteogenesis in radiation-induced bone injury. Specifically, radiation activates Fis1 expression, which induces excessive mitochondrial fission, leading to mitochondrial fragmentation, along with reduced capacities for oxidative phosphorylation, ATP synthesis, and antioxidant defense, that collectively impairs MSC osteogenesis and results in bone loss in radiation-induced bone injury. This process involves increased calcium (Ca²⁺) influx that stimulates calcineurin (CaN) to promote nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) dephosphorylation and nuclear translocation, which in turn, activates the transcriptional expression of Fis1. Consistent with the pivotal role of Fis1 in regulating mitochondrial fission and MSC osteogenesis, inhibition of Fis1 remarkably reduced mitochondrial fragmentation, enhanced MSC osteogenesis and reduced bone loss, highlighting the therapeutic potential of targeting Fis1 in radiation-induced bone injury. Our study provides new insights into the mechanisms and therapeutic strategies for radiation-induced bone injury.</p>