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Background: Juglone is a natural pigment, which has a cytotoxic effect against tumor cells. However, its cytotoxicity to human breast cancer cells (Michigan Cancer Foundation-7 [MCF7]) has not been demonstrated. Objective: The objective was to observe the effect of Juglone on the protein expression of caspase-3, 9 in the apoptosis of Human Breast Cancer Cells (MCF7) and apoptosis-related protein, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) and investigate in the inhibitory effect of MCF7. Materials and Methods: Methyl thiazolyl tetrazolium experiment was performed for the detection of the cell growth inhibition effect of MCF7. The apoptosis rate was detected by flow cytometry. Western Blot was used to detect the protein expression of Bax, Bcl-2, and caspase. The messenger RNA (mRNA) was tested using quantitative real-time polymerase chain reaction. Results: Juglone inhibited the growth of MCF7 cells in a concentration- and time-dependent manner and promotes the apoptosis of MCF7 cells in a concentration-dependent manner. Compared with control group, the Juglone group raised the expression of Bax protein (*P < 0.05), and the protein expression of Bcl-2 was decreased (*P < 0.05). The expression of caspase-3 protein was not changed, and the caspase-9 was significantly elevated in the high concentration of Juglone. Cleaved caspase-3, -9 protein was significantly raised (*P < 0.05). The mRNA levels of Bax, caspase-3, and caspase-9 in MCF7 cells of Juglone group were significantly increased compared with control, while the expression of Bcl-2 was suppressed obviously. Conclusion: Juglone inhibited the growth of MCF7 cells and promotes the apoptosis of MCF7 cells. Its mechanism in promoting MCF7 cell apoptosis may be related to the decrease of the expression of the mitochondrial pathway-associated apoptosis factor Bcl-2, increase of the protein expression of Bax, and mitochondrial pathway downstream caspase-3, 9. [ABSTRACT FROM AUTHOR]