Breast cancer ranks first among female cancers and has become a major public health problem in the current society. More studies indicated that these cancers are related to the change in the gut microbiome that can cause metabolic and immune system disorders in the body. However, there are few studies on the changes in gut microbiome caused by the onset of breast cancer, and the relationship between breast cancer and gut microbiome needs to be further clarified. In this study, we inoculated 4T1 breast cancer cells to induce breast cancer tumorigenesis in mice and collected their feces samples at different stages during this process. These intestinal florae were analyzed using 16S rRNA gene amplicon sequencing, and the results showed that at the phylum level, the ratio ofFirmicutes/Bacteroidetesdecreased with the development of the tumor; at the family level, the intestinal microbiome had obvious variations ofLachnospiraceae, Bacteroidaceae, Erysipelotrichaceae, etc. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and COG annotation demonstrated that decreased abundance of cancer-related signaling pathways. This study elucidated the relationship between breast cancer and intestinal microbiome, and the research results can be used as an important biomarker for the diagnosis of breast cancer.