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Background: Gut microbiota disorders have been closely related to polycystic ovarian syndrome (PCOS). Buzhong Yiqi prescription (BZYQ) has a great clinical effect on the treatment of obese patients with PCOS and phlegm-dampness syndrome caused by spleen deficiency (SPSD). This study was performed to explore the alterations in the gut microbiota and fecal metabolites in obese patients with PCOS and SPSD who received BZYQ treatment. Methods: A total of 50 obese patients with PCOS and SPSD were recruited from the Changhai Hospital in Shanghai and accepted three months of BZYQ treatment. Sex hormone were detected and oral glucose tolerance was tested in the outpatient laboratory before and after the BZYQ treatment. Fecal samples were detected by 16S rRNA high-throughput sequencing and nontargeted metabolomic methods to determine the structure of the gut microbiota and metabolites, respectively.Results: BZYQ could significantly alleviate the serum DHEAS (p<0.001) and T level (p<0.001) in obese patients with PCOS and SPSD. The structure of the gut microbiota changed significantly after the BZYQ treatment. In particular, at the phylum level, the abundance of Spirochaetae was significantly higher after treatment than that before treatment. At the genus level, the abundances of [Eubacterium]_rectale_group, Escherichia-Shigella, and Fusicatenibacter were significantly higher after treatment than those before treatment, but the abundance of Megamonas was significantly lower. A total of 106 differential metabolites and 14 KEGG enrichment pathways were quantified. The disorder in the gut microbiota and fecal metabolites of obese patients with PCOS and SPSD were closely related to hyperandrogenemia and insulin resistance. The level of tetracosanoic acid was negatively correlated with serum DHEA level (p<0.05), while the palmitoleic acid level was negatively correlated with serum T level (p<0.05). Conclusions: BZYQ could ameliorate the serum androgen level and had an impact on the gut microbiota and metabolites in obese patients with PCOS and SPSD.Trial registration: Chinese Clinical Trial Registry, ChiCTR-IPR-16009166. Registered 26 September 2016, http://www.chictr.org.cn/showproj.aspx?proj=14956