Abstract Estrogen, 17β-estradiol (E2) has multiple functions in breast cancers including stimulating cancer growth and interfering with chemotherapeutic efficacy. Heteronemin, a marine sesterterpenoid-type natural product, processes anti-proliferative effect in cancer cells. In the current study, we investigate the antitumor mechanism of heteronemin in breast cancer cells and further explore its molecular targets. Heteronemin exhibited potent cytotoxic effects against breast cancer cells. On the other hand, E2 stimulated cancer cell growth. Heteronemin and E2 showed different effects on gene expression of proliferation, angiogenesis or growth factor receptors in breast cancer cells. NanoString® analysis was further used to detect mRNAs, heteronemin repressed gene expression of transforming growth factor (TGF)-β, mothers against decapentaplegic homolog (SMAD) and PCNA, whose protein products play important roles in regulating cell growth, angiogenesis, and metastasis. The results indicate that heteronemin was able to modulate cell adhesion, expression of extracellular matrix (ECM) receptors, TGF-β pathway, cell motility, membrane integration, metastasis response, matrix metalloproteinase (MMP) remodeling, regulation of metabolism, sprouting angiogenesis, transcription factors, and vasculogenesis in breast cancer cells. However, those effects were partially reversed by E2. Furthermore, Heteronemin and E2 altered several signaling transduction pathways. In summary, this study provides insight into the complex pathways by which anti-proliferation is induced by heteronemin in E2-depleted and -repleted environments. Citation Format: Zi-Lin Li, Yi-shin Pan, Tung-Yung Huang, Ya-Jung Shih, Yi-Ru Chen, Heng-Yuan Tang, Jacqueline Whang-Peng, Hung-Yun Lin, Paul J. Davis, Kuan Wang. Effect of estrogen on heteronemin-induced anti-proliferation in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5034.