A diverse and large community of gut microbiota reside in the intestinal tract of various organisms and play important roles in metabolism and immune homeostasis of its host. The disorders of microbiota-host interaction have been closely associated with numerous chronic inflammatory and metabolic diseases, including inflammatory bowel disease and type 2 diabetes. The accumulating evidence has shown that fine particulate matter (PM_2.5) exposure contributes to the diabetes, atherosclerosis and inflammatory bowel diseases; however, few studies have explored the impact of inhaled diesel PM_2.5 on gut microbiota in vivo. In this study, C57BL/6J mice were exposed to diesel PM_2.5 for 14 days via intratracheal instillation, and colon tissues and feces were harvested for microbiota analysis. Using LC Sciences’ high-throughput sequencing technology, the researchers observed that intratracheally instillated diesel PM_2.5 significantly altered the gut microbiota diversity and community. At the phylum and genus levels, principal coordinate analysis (PCoA) and principal component analysis (PCA) indicated pronounced segregation of microbiota compositions, which were further confirmed by β diversity analysis. As the most affected phylum, Bacteroidetes was greatly diminished by diesel PM_2.5. On the genus level, Escherichia, Parabacteroides, Akkermansia, and Oscillibacter were significantly elevated by diesel PM_2.5 exposure. The findings provided clear evidence that exposure to diesel PM_2.5 via intratracheal instillation deteriorated the gastrointestinal (GI) tract and significantly altered the structure and composition of gut microbiota, which might subsequently contribute to the developmental abnormalities of inflammation, immunity and metabolism.