Objective: Citrus unshiu Markovich, which has been used for many different purposes in traditional medicine, has been reported to possess various pharmacological properties; however, its anticancer potentials are relatively unknown. This study aimed to investigate the effect of the ethanol extract of C. unshiu peel (EECU) on MCF-7 human breast cancer cells. Materials and Methods: Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected using DAPI staining and flow cytometry. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assay, caspase activity, and Western blotting analysis were used to confirm the basis of apoptosis. Results: Our results demonstrated that the inhibition of MCF-7 cell survival by EECU was associated with the induction of apoptosis. EECU-induced apoptosis resulted in a sequence of events, which began with the increased expression of death receptor-related proteins and a Bax/Bcl-2 expression ratio. This led to the collapse of MMP and the cytosolic release of cytochrome c, which was accompanied by and the activation of caspase-9 and caspase-8 and proteolytic degradation of poly (ADP-ribose) polymerase. EECU also induced apoptosis of MCF-7 cells by stimulating AMP-activated protein kinase (AMPK), through the generation of ROS. However, compound C, a pharmacological inhibitor of AMPK, significantly weakened EECU-induced apoptosis. Furthermore, the activation of AMKP, induction of apoptosis, and reduction of cell viability by EECU were effectively prevented when ROS production was blocked. Conclusions: These results demonstrate that EECU inhibits MCF-7 cell proliferation by activating the intrinsic and extrinsic apoptosis pathways through the ROS-dependent activation of the AMPK pathway.