Abstract Introduction: The microbiota is known to be involved in the development and progression of breast cancer, although few experiments have been conducted to determine which species in the microbiota are involved in the disease. We have examined the effects of symbiotic bacteria in breast cancer under hormone therapy and have identified bacteria that affect anti-hormonal therapy focusing on the estrogen receptor. The intestinal symbiotic bacteria secrete bacterial extracellular vesicles to the blood, lymphatic fluid, and communicate with distant organs through these vesicles which are including metabolites and bacterial materials. After circulation of the body, all the extracellular vesicles are collected to urine. Therefore, we analyzed the microbiome of breast cancer patients and normal controls through urine samples. Materials and Methods: A total of 347 urine samples from females (127 breast cancer patients and 220 normal individuals) were collected from September 2014 to August 2015, at Ewha Womans University Mokdong Hospital and Inje University Haeundae Paik Hospital. These samples analyzed by NGS using a universal bacterial primer of 16S rDNA. A t-test and Kaplan-Meier analysis were performed to find out the discrepancy between cancer and control groups. Cell experiments confirmed the effect of bacterial EVs on breast cancer cells. Results: We have found that there is a significant difference in the number of microorganisms between breast cancer patients and healthy controls. Among the microorganisms in breast cancer patients, a certain bacteria was found to be abundant in luminal A subtype tumors: Klebsiella. We selected Klebsiella pneumoniae for subsequent experiments. Over a mean 5-year follow-up period, The Klebsiella-abundant and -lacking groups included 50 and 48 patients, respectively, and the disease-free survival outcome of breast cancer patients was determined in both groups. Extracellular vesicles derived from Klebsiella showed anti-hormonal effects in MCF7 cells during treatment with tamoxifen. K. pneumoniae EVs doubled the efficacy of tamoxifen. K. pneumoniae EVs co-treated with tamoxifen down-regulated cyclin E2, which in turn decreased the expression of p21. Moreover, in this process, the ERK pathway appears to be involved. Conclusions: Based on clinical data and in vitro experiments with cell lines, we conclude that symbiotic microorganisms affect hormone therapy for breast cancer. These findings provide new horizons for breast cancer mechanisms and hormone therapy. Citation Format: Jeongshin An, Jong Bin Kim, Eun Yeol Yang, Jinho Yang, Won-Hee Lee, Hyungoo Kim, Jun Woo Lee, Joohyun Woo, Hyungju Kwon, Woosung Lim, Nam Sun Paik, Yoon-Keun Kim, Byung-In Moon. Urine microbiome and breast cancer: The relationship with hormone therapies [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-06.