Background. The incidence of small intestinal injury caused by low-dose aspirin (LDA) is high, but the pathogenesis and intervention measures of it have not been elucidated. Recent studies have found gut microbiota to be closely associated with onset and development of NSAID-induced intestinal injury. However, studies of the changes in the gut microbiota of rats with LDA-related intestinal injury have been lacking recently. In this study, we investigated fecal 16S rRNA gene sequencing analysis of changes in the gut microbiota of rats with LDA-related intestinal injury. Methods. Sprague-Dawley (SD) rat models of small intestinal injury were established by intragastric administration of LDA. The small intestinal tissues and the fecal samples were harvested. The fecal samples were then analyzed using high-throughput sequencing of 16S rRNA V3-V4 amplicons. The gut microbiota composition and diversity were analyzed and compared using principal coordinate analysis (PCoA), nonmetric multidimensional scaling (NMDS) analysis, the unweighted pair-group method with arithmetic mean (UPGMA) clustering analysis, multivariate statistical analysis (ANOSIM, MetaStats, and LEfSe), and spatial statistics. Results. The LDA rat model was successfully established. Decreased Firmicutes and increased Bacteroidetes abundances in rats with LDA-induced small intestinal injury were revealed. MetaStats analysis between the before administration of LDA (CG) and after administration of LDA (APC) groups showed that the intestinal floras exhibiting significant differences (P<0.05, q<0.1) were Firmicutes, Bacteroides, Cyanobacteria, Melainabacteria, Coriobacteriia, Bacteroidia, Bacteroidales, Eubacteriaceae, and Streptococcaceae. In addition, the bacterial taxa showing significant differences between the control (NS) and APC groups were Firmicutes, Bacteroides, Verrucomicrobiaceae and Peptococcaceae. Conclusions. The alterations in the gut microbiota composition and diversity of rats with LDA-related intestinal injury were found in the present study. The change of gut microbiota in LDA-related intestinal injury will lay the foundation for further research on the function and signaling pathways of the intestinal flora and promote the use of intestinal flora as drug targets to treat LDA-induced small intestinal injury. © 2021 Tianyu Chi et al.