Radiotherapy constitutes a pivotal approach in the management of pelvic malignant tumors. Despite notable strides in radiation technology, the incidence of collateral damage to neighboring normal tissues remains a prominent concern. Radiation-induced rectal injury (RRI) significantly impacts the postoperative quality of life of patients undergoing pelvic radiotherapy. We gathered RRI tissues alongside non-irradiated rectal tissues for a quantitative proteomic examination employing data-independent acquisition proteomics. Our analysis unveiled differentially expressed proteins (DEPs). Gene Ontology and Kyoto Encyclopedia of Genes and Genome enrichment analyses of DEPs uncovered conventional pathway modifications such as cytokine activation and extracellular matrix alterations, alongside emerging potential biological mechanisms implicated in RRI, including neutrophil extracellular traps, macrophage-to-myofibroblast transition, and metabolic reprogramming.