Tubeimoside I (TBMS1) is a natural triterpenoid saponin extracted from the rhizome of Bolbostemma paniculatum and has been proven to be an effective antitumor agent for the treatment of various human cancers. In this study, we used a comprehensive approach involving bioinformatics analysis and in vivo and in vitro experiments to investigate the effects of TBMS1 on triple-negative breast cancer (TNBC) cells and elucidate the underlying molecular mechanisms involved. In vitro experiments revealed a dose-dependent decrease in the growth, movement, and infiltration of MDA-MB-231 and MDA-MB-468 cells due to TBMS1. A greater increase in the TBMS1 concentration (0, 7.58, and 15.16 μM) was associated with a greater decrease. The flow cytometry results indicated that TBMS1 promoted apoptosis and induced cell cycle arrest in TNBC cells. The immunoblotting and NR3C2 gene knockdown results suggested that the inhibitory effect of TBMS1 on TNBC may be mediated via the NR3C2/PI3K/AKT signaling pathway. Comprehensive bioinformatics analysis was used to dissect the mechanism underlying the action of TBMS1 in TNBC cells at the molecular level. In the in vivo experiments, we established subcutaneous tumor xenograft models in female BALB/c mice, confirming the notable antitumor activity of TBMS1. The findings of our study demonstrate that TBMS1 significantly inhibits TNBC via the NR3C2/PI3K/AKT signaling pathway, suggesting that TBMS1 could be a potential tumor inhibitor.