© 2022, The Author(s), under exclusive licence to Springer Nature B.V.Background: The c-myc oncogene, which causes glutamine dependence in triple negative breast cancers (TNBC), is also the target of one of the signaling pathways affected by β-Escin. Methods and results: We sought to determine how c-myc protein affects glutamine metabolism and the proteins, glutamine transporter alanine-serine-cysteine 2 (ASCT2) and glutaminase (GLS1), in β-Escin-treated MDA-MB-231 cells using glutamine uptake and western blot analysis. Cell viability, colony formation, migration and apoptosis were also evaluated in MDA-MB-231 cells in response to β-Escin treatment using MTS, colony forming, wound healing, and Annexin-V assay. We determined that β-Escin decreased glutamine uptake and reduced c-myc and GLS1 protein expressions and increased the expression of ASCT2. In addition, this inhibition of glutamine metabolism decreased cell proliferation, colony formation and migration, and induced apoptosis. Conclusions: In this study, it was suggested that β-Escin inhibits glutamine metabolism via c-myc in MDA-MB-231 cells, and it is thought that as a result of interrupting the energy supply in these cells via c-myc, it results in a decrease in the carcinogenic properties of the cells. Consequently, β-Escin may be promising as a therapeutic agent for glutamine-dependent cancers.