Since dietary polyphenols can have beneficial effects in prevention and treatment of cancer, we tested the hypothesis that breast cancer patients’ intestinal microbiota is modulated by genistein (GE), an isoflavone found in soy, and that microbial alterations may offset the side effects brought about by chemotherapy. We demonstrated successful humanization of germ-free mice by transplanting fecal samples from breast cancer patients before and after chemotherapy. Mice were then grouped based on chemotherapy status and GE or control diet. We did not find any significant differences between pre-chemotherapy and post-chemotherapy bacterial composition and abundances. Germ-free mice on a GE diet showed differences in microbial composition as compared to mice on control diet. Four weeks after introduction of the customized GE diet, there was distinct clustering of GE-fed mice as compared to the control-fed group. In the gut microbiome of GE-treated humanized mice, there was an increase in abundance of genera Lactococcus and Eubacterium. Phylum Verrucomicrobia showed statistically significant (p = 0.02) differences in abundances between the GE-fed and control-fed groups. There was an increase in bacteria belonging to family Lachnospiraceae and Ruminococcaceae in GE-fed mice. Marked changes were observed in GE catabolism in mice humanized with fecal material from two of three patients’ post-chemotherapy with complete disappearance of 4-ethylphenol and 2-(4-hydroxyphenol) propionic acid conjugates. The post-tumor samples did not show any distinct clustering of the gut microbiota between the two diet groups. There was an increase in latency of about 25% for tumor growth of the humanized mice that were on a GE diet as compared to humanized mice on a control diet. The average tumor size for the GE group was significantly decreased compared to the non-GE group. Collectively, our results suggest that the intestinal microbiota becomes altered with a GE diet before induction of tumor. Our findings indicate that GE modulates the microbiome in humanized mice that may contribute to its effects on increasing the latency of breast tumor and reducing tumor growth.