Objective: To investigate the molecular mechanisms and pathway information of acute and chronic radiation enteritis following neoadjuvant radiotherapy for rectal cancer by proteomics-based approaches. Methods: Data-Independent Acquisition (DIA) proteomics was used to compare the non-tumor rectal tissues from patients without radiotherapy (group C) with those collected at 60~90 days (group E) and 150~160 days (group P) after radiotherapy. Differentially expressed proteins (DEPs) with |fold change (FC)| > 1.5 and P<0.05 were screened, and enrichment analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Groups (KOG) and protein domain were performed on these DEPs. Results: Compared with group C, 94 DEPs were up-regulated and 44 were down-regulated in group E, the up-regulated proteins were mainly enriched in peptide hormone processing, neuroactive ligand-receptor interaction pathway and synaptic protein domains. In the comparison of group P and group C, 46 DEPs were up-regulated and 56 were down-regulated, with the up-regulated proteins primarily enriched in negative regulation of DNA recombination, mitogen-activated protein kinase (MAPK) and Wingless-type (Wnt) signaling pathways, accompanied by changes in domains such as basic leucine zipper. Conclusion: The early phase of radiation-induced rectal injury is characterized by hyperactivity of the enteric neuro-endocrine system, whereas chronic inflammation and matrix deposition in the late phase are likely driven by the activation of MAPK/Wnt pathways. These findings may offer novel insights for the prevention and treatment of radiation enteritis.