Radiation enteritis (RE) is the most common complication of radiotherapy for pelvic irradiation receivers. Herein we investigated the alterations in gut microbial profiles and their association with enteritis in patients undergoing pelvic radiotherapy. Faecal samples were collected from 18 cervical cancer patients during radiotherapy. Microbiota profiles were characterized based on 16S rRNA sequencing using the Illumina HiSeq platform. Epithelial inflammatory response was evaluated using bacterial‐epithelial co‐cultures. Dysbiosis was observed among patients with RE, which was characterized by significantly reduced α‐diversity but increased β‐diversity, relative higher abundance of Proteobacteria and Gammaproteobacteria and lower abundance of Bacteroides . Coprococcus was clearly enriched prior to radiotherapy in patients who later developed RE. Metastat analysis further revealed unique grade‐related microbial features, such as more abundant Virgibacillus and Alcanivorax in patients with mild enteritis. Additionally, using bacterial‐epithelial co‐cultures, RE patient‐derived microbiota induced epithelial inflammation and barrier dysfunction, enhanced TNF‐α and IL‐1β expression compared with control microbiota. Taken together, we define the overall picture of gut microbiota in patients with RE. Our results suggest that dysbiosis of gut microbiota may contribute to development and progression of RE. Gut microbiota can offer a set of biomarkers for prediction, disease activity evaluation and treatment selection in RE.