Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. As yet, chemotherapy with drugs such as doxorubicin is the main treatment strategy. However, drug resistance and dose-dependent toxicities restrict their clinical use. Natural products are major sources of anti-tumor drugs. OSW-1 is a natural compound with strong anti-cancer effects in several types of cancer, but its effects on the efficacy of chemotherapy in TNBC and its underlying mechanism remain unclear.The inhibitory activities of OSW-1 and its combination with several chemotherapy drugs were tested using in vitro assays and in vivo subcutaneous and metastatic mouse TNBC models. The effects of the mono- and combination treatments on TNBC cell viability, apoptosis, autophagy and related signaling pathways were assessed using MTT, flow cytometry, RNA sequencing and immunology-based assays. In addition, the in vivo inhibitory effects of OSW-1 and (combined) chemotherapies were evaluated in subcutaneous and metastatic mouse tumor models.We found that OSW-1 induces Ca2+-dependent mitochondria-dependent intrinsic apoptosis and cyto-protective autophagy through the PI3K-Akt-mTOR pathway in TNBC cells in vitro. We also found that OSW-1 and doxorubicin exhibited strong synergistic anti-TNBC capabilities both in vivo and in vitro. Combination treatment strongly inhibited spontaneous and experimental lung metastases in 4T1 mouse models. In addition, the combination strategy of OSW-1 + Carboplatin + Docetaxel showed an excellent anti-metastatic effect in vivo.Our data revealed the mode of action and molecular mechanism underlying the effect of OSW-1 against TNBC, and provided a useful guidance for improving the sensitivity of TNBC cells to conventional chemotherapeutic drugs, which warrants further investigation.© 2022. Springer Nature Switzerland AG.