Breast cancer is one of the major causes of mortality among women. Due to many side effects of the existing chemotherapeutic agents, the research of anti-cancer drugs, including natural products, is still a big challenge. Here, we investigated the effects of colchicine on apoptosis of two breast cancer cell lines ( human MCF-7 and mouse 4T1). In this experimental study, we evaluated the apoptotic effects of colchicine on (MCF-7) and (4T1), as well as a human cancer-associated fibroblast cell line as a control group. Extraction and chromatographic techniques were applied to isolate colchicine from Colchicum autumnale L. To compare the isolated colchicine with pure standard colchicine, we used the H-NMR technique. The methyl thiazolyl tetrazolium (MTT) assay, quantitative reverse transcriptase-polymerase chain reaction, Western blotting and annexin V/PI staining were used to evaluate the apoptotic effects of the isolated and standard colchicine. Similar to standard colchicine, the isolated colchicine inhibited cell proliferation significantly in cancer cell lines. Colchine inhibited proliferation and induced apoptosis on a dose-dependent manner. The medicine modified the expression of genes-related to apoptosis by up-regulation of P53 ,BAX, CASPASE-3, -9 and down-regulation of BCL-2 gene, which led to an increase in the BAX/BCL-2 ratio. We showed that isolated colchicine from Colchicum autumnale and pure standard colchicines modulate the expression levels of several genes and therefore exerting their anticancer effects on both human (MCF-7) and mouse (4T1) breast cancer cells. Based on these results, we suggest that colchicine can be a potential candidate for prevention and treatment of breast cancer.