Objective To investigate the effects of the estrogen synthesis inhibitor letrozole on the composition, diversity, and abundance of the intestinal microbiota in rats with polycystic ovary syndrome (PCOS). Methods A rat model of PCOS was induced using the estrogen synthesis inhibitor letrozole. The gut microbiota structures of normal and PCOS rats were analyzed 16S rRNA sequencing. Results At the phylum level, rats in the PCOS group had a lower abundance of Firmicutes (73. 29% ± 5. 34% vs. 77. 17% ± 4. 01%) and a higher abundance of Bacteroidetes (23. 85% ± 6. 22% vs. 17. 21% ± 6. 90%) compared with the normal group. At the genus level, the abundances of Rochella (5. 61% ± 3. 20% vs. 16. 13% ± 6. 20%) and Zurichella (1. 75% ± 2. 23% vs. 9. 98% ± 7. 34%) were lower in the PCOS group than in the normal group, but the abundances of Lactobacillus (18. 83% ± 11. 50% vs. 15. 88% ± 7. 06%), Prevotella(4. 58% ± 1. 09% vs. 0. 46% ± 0. 15%), and Ruminococcus (2. 97% ± 1. 56% vs. 1. 16% ± 0. 60%) were increased.Analysis of Kyoto Encyclopedia of Genes and Genomes signaling pathways of the differentially expressed bacteria showed that signaling pathways related to bile acid biosynthesis in the intestine were changed in the PCOS rat model. Conclusions The structure of intestinal flora in PCOS rat model constructed by intragastric administration of letrozole is significantly disordered, which may be related to the signaling pathways related to bile acid biosynthesis.