OBJECTIVES: Enrichment for therapy-resistant cancer stem cells hampers the treatment of triple-negative breast cancer. Targeting these cells via suppression of Notch signalling can be a potential therapeutic strategy. This study aimed to uncover the mode of action of a new indolocarbazole alkaloid loonamycin A against this incurable disease. METHODS: The anticancer effects were examined in triple-negative breast cancer cells using in vitro methods, including cell viability and proliferation assays, wound-healing assay, flow cytometry and mammosphere formation assay. RNA-seq technology was used to analyse the gene expression profiles in loonamycin A-treated cells. Real-time RT-PCR and western blot were to evaluate the inhibition of Notch signalling. KEY FINDINGS: Loonamycin A has stronger cytotoxicity than its structural analog rebeccamycin. Besides inhibiting cell proliferation and migration, loonamycin A reduced CD44high/CD24low/- sub-population, mammosphere formation, as well as the expression of stemness-associated genes. Co-administration of loonamycin A enhanced antitumour effects of paclitaxel by inducing apoptosis. RNA sequencing results showed that loonamycin A treatment caused the inhibition of Notch signalling, accompanied by the decreased expression of Notch1 and its targeted genes. CONCLUSIONS: These results reveal a novel bioactivity of indolocarbazole-type alkaloids and provide a promising Notch-inhibiting small molecular candidate for triple-negative breast cancer therapy. CI - (c) The Author(s) 2023. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.