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AIM The use of plant-derived drugs in cancer therapy is widely considered in the treatment of different malignancies including breast cancer. Cucurbitacin D (CuD) is able to induce apoptosis in cancerous cells through different signaling pathways. The aim of this study was to examine the effect of different concentrations of CuD on viability and death pattern. METHODS Antiproliferative effects of CuD on these cell lines' viability were investigated using the MTT assay. Real-time PCR was applied to evaluate the expression alterations of Bcl-2, Bax, caspase-3, p53 (that related to apoptotic cell death pathway), Atg5, Beclin-1, PTEN, and Akt genes (autophagy genes) in the MCF-7 (ER positive) and MDA-MB-468 (triple negative) breast cancer cells. RESULTS Significant dose-dependent and antiproliferative effects of CuD were observed on MCF-7 and MDA-MB-468 cells after 24 h with IC50 value about 30 and 25 µM, respectively (p < 0.01). Significant changes in expression of the genes in the breast cancer lines were observed under different concentrations of CuD. CONCLUSION Our results confirmed that CuD may influence breast cancer cell lines' viability at specific doses and by altering the expression of these genes. The differences between the gene's aberrations in our breast cancer cell lines propose that these genes can have a distinct role in the pathophysiology and therapy responsiveness of various subtypes of breast cancer.