Background: Grape seed extract is a complex mixture of polyphenols. Its anti-tumor effects have been reported by several studies. Estrogen receptors (ERs) are commonly considered as important markers for breast cancer. The present study aimed to evaluate the apoptotic effects of GSE on MCF7 breast cancer cells and assessed the expression of ERβ during treatment of cells with GSE. Material and Methods: The half maximal inhibitory concentration (IC 50 ) of GSE in MCF7 breast cancer cells were calculated by treating cells with serial dilution of GSE for 48 hours and cell viability evaluated using MTT assay. Then cells assigned to three groups: control (no treatment), DMSO (cells treated with 0.05% of DMSO) and GSE group (cells treated with of GSE for 48 hours). The apoptosis assay was performed by detecting Annexin V protein by flow cytometry. The gene expression of ERβ and caspase-3 was evaluated by Real-Time PCR. Results: Cells in GSE group treated with GSE IC 50 concentration for 48 hours. Annexin V staining assay, represented early apoptosis detected by flow cytometry analysis showed significantly higher expression (p<0.01) than control and DMSO groups. Moreover, results of Real-Time PCR showed a significant expression in ERβ and caspase-3 genes in GSE group compared to control and DMSO groups (Fold change = 2.3 and 3.5, respectively). Conclusion: GSE may induce apoptosis in MCF7 human breast cancer cells by activation of ERβ gene.