Abstract: The purpose of this study was to investigate the effects of long-distance transportation on the intestinal microbiota of horses using Illumina Mi Seq sequencing technology. In this experiment, anal swabs were collected from horses（Equus） at 3 time points: before transport, 16 h during transport, 16 h after transport. The results showed that transport stress had a significant effect on the diversity and composition of intestinal microbiota in horses.Alpha diversity showed that the species abundance was significantly higher at 16 h after transportation than that before transportation（P＜0.05）. Transportation has a different degree of influence on the analysis of the composition of the dominant bacteria in the horse intestinal flora at different classification levels. At the phylum level, Firmicutes, Bacteroidetes and Proteobacteria are the dominant phylums. After transportation, the average content of Firmicutes and Proteobacteria decreased, while the average content of Bacteroidetes increased. At the genus level,Streptococcus and Escherichia-shigella are the two dominant bacteria genera. After transportation, the average content of Streptococcus increased while that of Escherichia-shigella decreased. In the comparative analysis of species composition differences, the species composition differences were not significant at the phylum level. At the genus level, the average content of Christensenellaceae＿R-7＿group after transportation was 1.7%, which was significantly lower than that before transportation（2.87%）（P＜0.05）. The results of this study show that transportation stress can cause changes in the intestinal microbiota of horses, which can provide a basis for further research on the impact of horse transportation stress on the intestinal microbiota, and provide a reference for studying how to alleviate the changes of intestinal microbiota caused by horse transportation stress.