BACKGROUND Intestinal microbiota play a critical role in the development of colorectal cancer. However, little is known about the structure and characteristics of gut microbial in colorectal cancer, especially in locally advanced rectal cancer after neoadjuvant chemoradiation therapy. METHODS Here, we performed this study to evaluate microbial characteristics between pathologic complete response (pCR) (n=12) and non-pathological complete response (Non-pCR) (n=45) tumor tissues from patients with locally advanced rectal cancer after neoadjuvant chemoradiation therapy. In this study, 16S rRNA gene sequencing was used to detect the microbial diversity including Alpha diversity and Beta diversity. Moreover, we used PICRUSt from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to predict the microbial metabolism functions. RESULTS There was significant statistical difference in PFS between pCR and Non-pCR group (p < 0.05). However, there was no significant difference in OS between pCR and Non-pCR group. The microbial compositions in the both groups were Proteobacteria, Actinobacteria, Firmicutes and Thermi and Bacteroidetes at the phylum level. The five most predominant genera in both pCR and Non-pCR tissue groups were Sphingobium, Acinetobacter, Cupriavidus, Thermi and Sphingomonas at the genus level. The key taxa identified in the pCR and Non-pCR tissues were Thermi and Sphingomonadaceae respectively. In addition, a series of human disease-related genes were also significantly different between pCR and Non-pCR group. CONCLUSIONS In summary, we demonstrated the characteristic differences in microbial communities between pCR tissues and Non-pCR tumor tissues from locally advanced rectal cancer patients after neoadjuvant chemoradiation therapy. Our results present new alterations in the microbiome in locally advanced rectal cancer after neoadjuvant chemoradiation therapy, suggesting that it will provide a new perspective for the precise treatment of neoadjuvant rectal cancer by targeting specific microbial species in the future.