Background: Breast cancer (BC) is known as the most common cancer in women. Discovering novel and effective drugs is a priority for the treatment of BC. Oxypalmatine (OPT) is a natural protoberberine-type alkaloid isolated from Phellodendron amurense Rupr. (Rutaceae) with potential anti-cancer activity. Purpose: This investigation aimed to elucidate the biological role and potential mechanisms of OPT in BC cells, and intended to assess the therapeutic potential of OPT in BC patient-derived organoid models. Methods: CCK-8 and EdU assays, and flow cytometry were used to test the activity of OPT against BC cells. In addition, patient-derived organoid models were constructed to assess the therapeutic efficiency of OPT in BC. Besides, network pharmacological analysis and RNA sequencing analysis were performed to predict the under-lying anti-BC mechanism of OPT. Moreover, Western blot analysis was applied to test the expression of genes modulated by OPT. Results: OPT attenuated the proliferation and DNA replication, and induced apoptosis in multiple BC cells. Interestingly, OPT also exerted a cytotoxic effect on BC organoids characterized as luminal A, HER2-overexpressing, and triple-negative subtypes, indicating that OPT was a potential broad-spectrum anticancer drug. Network pharmacological analysis suggested that OPT might affect signals contributing to BC progression, including PI3K/AKT, MAPK, and VEGFA-VEGFR2 signaling pathways. Moreover, bioinformatics analysis of data from our RNA sequencing suggested that PI3K/AKT was a downstream pathway of OPT in BC. Finally, OPT was shown to inactivate PI3K/AKT signaling pathway in BC cells by Western blot analysis. Conclusions: Collectively, our study demonstrated that OPT suppressed proliferation and induced apoptosis through mitigating the PI3K/AKT signaling pathway in BC cells. Moreover, our work first adopted BC organoid models to confirm OPT as an effective and promising drug, laying a foundation for the potential use of OPT in BC treatment.