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Background Acute radiation-induced intestinal injury (ARII) is a common side effect of radiotherapy. While probiotics offer potential relief, conventional strains often fail due to poor colonization in the irradiated gut. This study aimed to identify novel probiotics with superior colonization and therapeutic efficacy.Methods We analyzed the gut microbiota of radiotherapy responders (patients achieving complete tumor remission without ARII) versus non- responders. A candidate strain, Lacticaseibacillus casei CY01, was isolated and evaluated for acid resistance, colonization stability post-irradiation, and its protective effects against ARII in vivo.Results Lacticaseibacillus species were identified as the key microbial signature differentiating responders from other patients. The newly isolated strain, CY01, demonstrated exceptional acid tolerance and sustained high colonization levels in the gut environment following radiation. Administration of CY01 significantly alleviated symptoms of ARII and improved intestinal barrier integrity in animal models with a high safety profile.Conclusions L. casei CY01 is a promising probiotic candidate derived from clinical responders, offering a robust and stable strategy for the prevention and treatment of radiation-induced intestinal damage.