Traditional medicine uses Annona muricata to treat a variety of illnesses. This study aimed to assess the anti-proliferative effects of the hexane fraction of Annona muricata (HFAM) seed in MCF-7 cells and 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary gland tumorigenesis in female rats. Forty Wistar rats were divided into five equal groups in vivo. The control group was group 1, the DMBA (50 mg/kg) was assigned to group 2, the DMBA (50 mg/kg) + HFAM (100 mg/kg) was given to group 3, the DMBA (50 mg/kg) + HFAM (200 mg/kg) was given to group 4, and the HFAM (200 mg/kg) was given to group 5. The HFAM inhibited growth, elicited anti-inflammatory, antioxidative, and pro-apoptotic activities in MCF-cells. The HFAM decreased IL-1β, MPO, LPO and increased SOD, CAT, BAX, Caspases-3 and-9 in MCF-7 cells by 45%, 82%, 46%, 44%, 41%, 168%, 22% and 17%, respectively. In vivo, DMBA decreased body weight gain and increased organo-somatic weight of the mammary gland by 35% and 92%, respectively. Also, DMBA decreased the activities of mammary catalase, glutathione-s-transferase, glutathione peroxidase, and superoxide dismutase by 40%, 66%, 45% and 41%, respectively, while lipid peroxidation increased by 61%. The GC-MS analysis revealed Tirucallol as the most abundant compound. Histology showed glands with malignant epithelial cells and high nucleocytoplasmic in DMBA-administered rats. Interestingly, HFAM decreased inflammation, oxidative stress, and restored the cyto-architecture of glands in DMBA-treated rats. Taken together, HFAM confers protection against DMBA-induced mammary toxicity via anti-inflammatory, antioxidative, and pro-apoptotic activities.