Objective: To investigate whether letrozole (an estrogen synthesis inhibitor) can affect the composition, diversity and abundance of intestinal microbiota in patients with polycystic ovary syndrome (PCOS). Methods: The PCOS rat model was induced and constructed by letrozole, an estrogen synthesis inhibitor. The gut microbiota structure of normal and PCOS rats was analyzed by 16S rRNA sequencing. Results: At the phylum level, the PCOS group had a lower abundance of Firmicutes (77.2% vs. 73.3%) and a higher abundance of Bacteroidetes (17.2% vs. 23.9%) than the normal group. At the genus level, the abundance of Rochella (16.1% vs. 5.6%) and Zurichella (10.0% vs. 1.8%) in PCOS group was lower than that in the normal group. However, the abundance of Lactobacillus (15.9% vs. 18.8%)、 Prevotella (0.5% vs. 4.6%) and Ruminococcus (1.2% vs. 3.0%) increased. The KEGG signaling pathways of the two groups of differentially expressed bacteria were further analyzed. The results showed that the signaling pathways related to bile acid biosynthesis in the intestine were changed in the PCOS rat model. Conclusion: These results provide a basis for further in-depth study of microbial structure and function and regulatory mechanisms in PCOS patients.