Background <p>Endometrial cancer (EC) is one of the most common malignant tumors in women, and in recent years, the role of gut microbiota in tumorigenesis has gradually gained attention. Previous studies have shown that the gut microbiome is closely related to the occurrence of various cancers, but the specific mechanisms through which gut microbiota contribute to the development of endometrial cancer (EC) remain unclear. This study aims to analyze the gut microbiome characteristics of atypical endometrial hyperplasia (AEH) and EC, and to explore key gut microbial species and metabolites, providing evidence for the etiological research and early screening of EC and AEH.</p> Methods <p>This study selected 24 AEH or EC patients from the Gynecology Department of Gansu Provincial Maternity and Child Care Hospital between February 2023 and October 2023. The patients were divided into the AEH group (n=7) and the EC group (n=17), with 24 healthy women selected as a control group. Fecal and serum samples were collected, and 16S rRNA gene sequencing was performed using the Illumina MiSeq platform. Serum metabolomics analysis was conducted using LC-MS technology. Spearman correlation analysis was used to explore the associations between gut microbiota and metabolites, and potential gut microbial biomarkers were evaluated using ROC analysis.</p> Results <p>The study found that as AEH progressed to EC, significant changes occurred in the composition of the gut microbiota, particularly in Klebsiella, whose abundance increased from 0.264% in the control group to 0.809% in the AEH group and 6.092% in the EC group, with significant differences (P&lt;0.001, FDR=0.026). LEfSe analysis identified Megamonas, Klebsiella, Escherichia, and Akkermansia as potential biomarkers. ROC analysis showed that the AUCs of Megamonas/Klebsiella for EC were 0.864/0.838, and the AUCs of Escherichia/Akkermansia for AEH were 0.744/0.920. Metabolomics analysis revealed significant enrichment of glycerophospholipid metabolism in both the EC and AEH groups, with significant differences in lipid metabolism in the EC group. Correlation analysis indicated significant positive correlations between Enterococcus and hypoxanthine, inosine in the EC group (r=0.686, 0.637, P&lt;0.05).</p> Conclusion <p>This study reveals the dynamic changes in the gut microbiome during the development of endometrial lesions, especially the increasing abundance of Klebsiella as AEH progresses to EC, suggesting that it may play a key role in the occurrence and progression of EC. Furthermore, significant changes in lipid metabolism further support the role of gut microbiota in regulating lipid metabolism in EC pathogenesis. This study provides new insights into the role of gut microbiota in endometrial cancer and offers a theoretical basis for early diagnosis and personalized treatment strategies based on gut microbiota.</p>