Background AG8, a triterpenoid saponin isolated from Ardisia gigantifolia Stapf., our previous studies found that AG8 inhibited the proliferation of triple negative breast cancers (TNBCs) by including ROS generation and triggering mitochondrial apoptotic pathways. Cancer stem cells (CSCs) capable of maintain relatively low levels of ROS, are vulnerable to the interference of redox state. Compounds capable of generating ROS can affect the finely balanced redox state of CSCs and have potency to selectively kill them. Objective We selected BT-549 which is most sensitive to AG8 to further study the effects of AG8 on hypoxia-induced proliferation, metastasis and CSCs. Methods The hypoxia condition was simulated by CoCl2 and cell viability assay of BT-549 cells was performed using MTT. Stemness phenotype were identified using breast CSCs marker (CD24?/low/CD44+) and mammosphere formation assay. Cell motility was determined via the wound healing assay and transwell migration. Protein levels of HIF1-α, Oct-4, SOX-2, c-MYC were tested by western blotting. Results In this study, AG8 showed significant cytotoxic effects on BT-549 triple negative breast cancer cells, but the effects were impaired by HIF1 activation. AG8 inhibited the hypoxia-induced migration and invasion of BT-549 cells. Further studies revealed that hypoxia-induced increases of CD44+CD24?/low and mammosphere population were significantly inhibited by AG8 dose-dependently. Moreover, AG8 decreased the Oct-4 and SOX-2 protein expression without affecting HIF1-α and c-MYC. AG8 significantly inhibited BT-549 xenograft tumors growth in BALB/c nude mice comparing with that of the control group. Conclusion In summary, AG8 inhibited hypoxia-induced cell migration and invasion through stemness regulations, indicating novel mechanisms for the antitumor effects of AG8 against triple negative breast cancer.