Polycystic ovary syndrome (PCOS) is a common disease of endocrine–metabolic disorder, and its etiology remains largely unknown. The gut microbiota is possibly involved in PCOS, while the association remains unclear. The comprehensive analysis combining gut microbiota with PCOS typical symptoms was performed to analyze the role of gut microbiota in PCOS in this study. The clinical patients and letrozole-induced animal models were determined on PCOS indexes and gut microbiota, and fecal microbiota transplantation (FMT) was conducted. Results indicated that the animal models displayed typical PCOS symptoms, including disordered estrous cycles, elevated testosterone levels, and ovarian morphological change; meanwhile, the symptoms were improved after FMT. Furthermore, the microbial diversity exhibited disordered, and the abundance of the genus Ruminococcus and Lactobacillus showed a consistent trend in PCOS rats and patients. The microbiota diversity and several key genera were restored subjected to FMT, and correlation analysis also supported relevant conclusions. Moreover, LEfSe analysis showed that Gemmiger, Flexispira, and Eubacterium were overrepresented in PCOS groups. Overall, the results indicate the involvement of gut microbiota in PCOS and its possible alleviation of endocrinal and reproductive dysfunctions through several special bacteria taxa, which can function as the biomarker or potential target for diagnosis and treatment. These results can provide the new insights for treatment and prevention strategies of PCOS.