Aconitine linoleate (1) is a lipo-diterpenoid alkaloid, isolated from Aconitum sinchiangense W. T. Wang. The study aimed at investigating the anti-proliferative efficacy and the underlying mechanisms of 1 against MCF-7 and MCF-7/ADR cells, as well as obvious the safety evaluation in vivo. The cytotoxic activities of 1 were measured in vitro. Also, we investigated the latent mechanism of 1 by cell cycle analysis in MCF-7/ADR cells and topo I and topo IIalpha inhibition assay. Molecular docking is done by Discovery Studio 3.5 and Autodock vina 1.1.2. Finally, the acute toxicity of 1 was detected on mice. 1 exhibited significant antitumor activity against both MCF-7 and MCF-7/ADR cells, with IC(50) values of 7.58 and 7.02 muM, which is 2.38 times and 5.05 times more active, respectively than etoposide in both cell lines, and being 9.63 times more active than Adriamycin in MCF-7/ADR cell lines. The molecular docking and the topo inhibition test found that it is a selective inhibitor of topoisomerase IIalpha. Moreover, activation of the damage response pathway of the DNA leads to cell cycle arrest at the G(0)G(1) phase. Furthermore, the in vivo acute toxicity of 1 in mice displayed lower toxicity than aconitine, with LD(50) of 2.2 x 10(5) nmol/kg and only slight pathological changes in liver and lung tissue, 489 times safer than aconitine. In conclusion, compared with aconitine, 1 has more significant anti-proliferative activity against MCF-7 and MCF-7/ADR cells and greatly reduces in vivo toxicity, which suggests this kind of lipo-alkaloids is powerful and promising antitumor compounds for breast cancer. CI - (c) 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.