Breast cancer is the second most common cause of cancer-associated mortality among women worldwide, and triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Berbamine (BBM) is a traditional Chinese medicine used for the treatment of leukopenia without any obvious side effects. Recent reports found that BBM has anti-cancer effects. The present study aimed to investigate the effects of BBM on TNBC cell lines and the underlying molecular mechanism. MDA-MB-231 cells and MCF-7 cells, two TNBC cell lines, were treated with various concentrations of BBM. A series of bioassays including MTT, colony formation, EdU staining, apoptosis, trypan blue dye, wound healing, transwell, ELISA and western blotting assays were performed. The results showed that BBM significantly inhibited cell proliferation of MDA-MB-231 cells (P<0.05; IC=22.72 μM) and MCF-7 cells (P<0.05; IC=20.92 μM). BBM (20 μM) decreased the apoptosis ratio (percentage of absorbance compared with the control group) by 28.4±3.3% (P<0.05) in MDA-MB-231 cells, and 62.4±24.6% (P<0.05) in MCF-7 cells. In addition, BBM inhibited cell migration and invasion of TNBC cells. Furthermore, the expression levels of PI3K, phosphorylated-Akt/Akt, COX-2, LOX, MDM2 and mTOR were downregulated by BBM, and the expression of p53 was upregulated by BBM. These results indicated that BBM may suppress the development of TNBC via regulation of the PI3K/Akt/MDM2/p53 and PI3K/Akt/mTOR signal pathways. Therefore, BBM might be used as a drug candidate for the treatment of TNBC in the future.