Background and Aims: Chemotherapy is the main treatment for metastatic triple negative breast cancer (mTNBC) but many patients develop resistance and progress to metastatic disease, with a 5-year survival rate of 11%. Therefore, more effective and less toxic treatments are required. 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3) are two pharmacologically active enantiomers extracted from Panax ginseng. In our studies, we have shown that these two enantiomers have stereoselective activities in inhibition of proliferation, migration and invasion of triple-negative breast cancer (TNBC) cells, and hence, could be considered as two separate drugs that can be combined in a synergistic dose (S+R). Based on the inhibitory effects of single enantiomers on loop formation of human umbilical vein endothelial cells (HUVEC), we have optimized the concentrations of Rg3 enantiomers to be combined that have shown time- and dose-dependent inhibition of loop formation and migration of human and mouse endothelial cell lines. The aim of the current study was to show the mechanisms of S+R in inhibition of angiogenesis in vitro and its efficacy in inhibiting metastasis in vivo. Methodology: Molecular docking was used to study the Rg3- vascular endothelial growth factor receptor (VEGFR) 2 interaction, and the VEGF bioassay kit (Promega) was used to show this interaction in vitro. ELISA, quantitative-PCR and western blot (WB) were used to study the expression of transcript and protein levels of signalling molecules. Two TNBC cell lines (MDA-MB-231 and HCC1143) were cultured as mammospheres and the effects of S+R on formation of mammospheres, viability (trypan blue assay and flow cytometric analysis of apoptosis or cell cycle arrest) and expression of CD44+ cells (flow cytometry) were studied. Expression microarrays were used to determine the effect of the treatment on the PI3K signalling pathway in MDA-MB-231. Nod Scid gamma mice were used to develop a murine model of metastatic breast cancer model. Luciferase-tagged MDA-MB-231 cells were injected into the 4th mammary fat pad of the mice and upon primary tumour establishment, treatment started. Tumour growth and metastasis was monitored using IVIS Spectrum. Statistical analysis was performed using ANOVA (Prism v8). Results: According to molecular docking, binding score of Rg3-VEGFR2 was -9.0 cal/mol. The VEGF bioassay showed that Rg3 is an allosteric modulator of VEGFR2 receptor. In HUVEC, S+R treatment decreased the expression of VEGF (ELISA) and its receptor VEGFR2. It also decreased the expression of pAKT:AKT, aquaporin 1 (AQP1) and pFAK:FAK (WB). These proteins are involved in cell survival, migration, invasion and proliferation. In breast cancer cell lines, S+R decreased the expression of AQP1 and decreased mammosphere formation efficiency, not by decreasing cell viability but by decreasing the expression of CD44+ stem cell marker. In expression arrays, the treatment decreased the expression of BTK, a tyrosine kinase promoting cell death escape; increased the expression of both CASP9, promoting apoptosis, and INPPD5, favouring inhibition of metastasis. In vivo experiments showed that S+R lead to tumour shrinkage (p < .0001), decreased body burden of tumour (p < .0001) and decreased number of metastases (p = .002). Conclusion: These results support the anti-cancer effects of the combination of S+R for the treatment of mTNBC. Citation Format: Maryam Nakhjavani, Eric Smith, Timothy Price, Amanda R Townsend, Jennifer E Hardingham. Ginsenoside rg3 enantiomers in a defined ratio as a novel treatment for metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-38.