Aim: The opening of mitochondrial permeability transition (mPT) pore is an important event in the execution of mitochondrial-mediated apoptosis. Some bioactive compounds elicit their chemotherapeutic effects against tumor/cancer cells via the induction of mitochondrial-mediated apoptosis. Annona muricata, a medicinal plant, is folklorically used in the treatment of tumors and cancers. This study therefore aimed at investigating the effect of methanol stem bark extract of Annona muricata (MEAM) on apoptosis via mPT pore and estradiol benzoate (EB)-induced proliferative disorder using female Wistar rats. Methodology: Mitochondria were isolated using differential centrifugation. The mPT pore opening, cytochrome c release and mitochondrial ATPase activity were determined spectrophotometrically. The levels of estrogen (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), malondialdehyde (MDA) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH), were determined using ELISA technique. Histological and histochemical assessments of the uterine sections were carried out using standard methods. Phytochemical constituents of MEAM were determined using Gas Chromatography-Mass Spectroscopy (GC-MS). Results: The in vitro results showed a significant induction of mPT pore opening, release of cytochrome c and enhancement of mitochondrial ATPase (mATPase) activity in a concentration-dependent manner. However, oral administration of MEAM did not induce rat uterine mPT pore opening, neither any significant release of cytochrome c nor enhancement of mATPase activity at the dosages used. Interestingly, MEAM reversed the EB-induced increase in E2, LH and FSH. The MEAM also improved the antioxidant milleu by reducing MDA level and increasing the SOD and GSH-Px activities in the treatment groups. Administration of EB induced endometrial hyperplasia in the model group which was mitigated by MEAM in the treatment group. The GC-MS analysis of MEAM revealed the presence of some important phytochemicals that are pharmacological relevant in cancer treatment. Conclusions: This study suggests that the methanol stem bark extract of Annona muricata contains bioactive compounds that protect against EB-induced uterine proliferative disorder in female Wistar rats.