This study determined the chemosensitizing potential of ginsenoside Rg1 in triple-negative MDA-MB-231 breast cancer cell lines. Ginsenoside Rg1 (10 µM) treated breast cancer cells were exposed to 8 nM of doxorubicin, and the chemosensitizing potential was measured by cell-based assays. Ginsenoside Rg1 (10 µM) treatment lowered the doxorubicin IC50 value to 0.01 nM. Furthermore, the ginsenoside pretreatment augments doxorubicin-mediated reactive oxygen species (ROS) generation and subsequent alterations of mitochondrial membrane potential in MDA-MB-231 cell lines. The alkaline comet assay results illustrated an increased % tail DNA during ginsenoside Rg1 plus doxorubicin treatment than doxorubicin alone treatment. In addition, the number of apoptotic cells was also increased in ginsenoside Rg1 plus doxorubicin-treated cells. Furthermore, the polymerase chain reaction array results illustrate activation of mitogen-activated protein kinase (MAPK) gene expression (AKT, ERK, and MAPK) during doxorubicin alone treatment and it has been attenuated by ginsenoside Rg1 pretreatment. Moreover, ginsenoside Rg1 treatment before doxorubicin activates the DNA damage response elements (ATM, H2AX, RAD51, and XRCC1) and subsequent apoptosis-related gene expression (p21, TP53. APAF1, Bax, CASP3, and CASP9) patterns in MDA-MB-231 cell lines. The ginsenoside Rg1 plus doxorubicin combination shows less cytotoxicity and ROS generation in MDA10A normal breast cancer cell lines. Therefore, the present results support the chemosensitizing property of ginsenoside Rg1 in triple-negative breast cancer cell lines.© 2021 Wiley Periodicals LLC.