Metabolic diseases like obesity, diabetes, and hypertension are considered major risk factors associated with endometrial cancer. Considering that an imbalance in the gut microbiome may lead to metabolic alterations, we hypothesized that alteration in the gut microbioma might be an indirect factor in the development of endometrial cancer. Our aim was to profile the gut microbiota of patients with endometrial cancer compared with healthy controls in this study. Thus, we used 16S rRNA high-throughput gene sequencing on the Illumina NovaSeq platform to profile microbial communities. Fecal samples were collected from 33 endometrial cancer patients (EC group) and 32 healthy controls (N group) between February 2021 and July 2021. The total numbers of operational taxonomic units (OTUs) in the N and EC groups were 28,537 and 18,465, respectively, while the number of OTUs shared by the two groups was 4771. This study was the first to report that the alpha diversity of the gut microbiota was significantly reduced in endometrial cancer patients vs. healthy controls. Also, there was a significant difference in the distribution of microbiome between the two groups: the abundance ofFirmicutes,Clostridia,Clostridiales,Ruminococcaceae, Faecalibacterium, andGemmiger_formicisdecreased, while that ofProteobacteria,Gammaproteobacteria,Enterobacteriales,EnterobacteriaceaeandShigellaincreased significantly in the EC group vs. healthy controls (allp < 0.05). The predominant intestinal microbiota of the endometrial cancer patients wasProteobacteria,Gammaproteobacteria,Enterobacteriales,Enterobacteriaceae, andShigella. These results imply that adjusting the composition of the gut microbiota and maintaining microbiota homeostasis may be an effective strategy for preventing and treating endometrial cancer.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00284-023-03361-6.