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Background Radioresistance limits radiotherapy (RT) efficacy in non-small cell lung cancer (NSCLC). Emerging evidence suggests that gut microbiota influence tumor responses to therapy, yet their role in RT sensitivity remains unclear. We investigated whether the butyrate-producing bacterium Clostridium butyricum enhances RT response in NSCLC and explored the underlying mechanisms.Methods Metagenomic sequencing compared the gut microbiota between NSCLC patients with and without RT response. Orthotopic and subcutaneous mouse models with antibiotic-mediated microbiota depletion and fecal microbiota transplantation were used to assess microbiota-dependent radiosensitivity. Metabolomic analysis identified key metabolites. The effects of Clostridium butyricum and butyrate on NSCLC radiosensitivity were evaluated in NSCLC cell lines and mouse models with irradiation. Proliferation, apoptosis, and DNA damage were analyzed. RNA sequencing was performed to evaluate the underlying mechanism.Results Microbiota depletion impaired, whereas fecal microbiota transplantation restored RT efficacy in NSCLC mouse models. Metagenomic analysis revealed enrichment of Clostridium butyricum in RT responders, which was confirmed in mouse models. Clostridium butyricum enhanced RT efficacy in orthotopic and subcutaneous tumors and increased radiosensitivity of NSCLC cell lines by suppressing proliferation and promoting apoptosis. Metabolomics identified butyrate as the key mediator enhancing RT response in vivo and in vitro through the gut-lung axis. Mechanistically, butyrate activated GPR109A-FOXO1 signaling and suppressed fatty acid oxidation (FAO), leading to metabolic reprogramming and increased radiosensitivity.Conclusions Clostridium butyricum-derived butyrate enhances radiosensitivity in NSCLC via the gut-lung GPR109A-FOXO1-FAO axis. Supplementation of butyrate-producing bacteria represents a potential strategy to overcome radioresistance in NSCLC.