Breast cancer is the most prevalent cancer and the second leading cause of cancer-related mortality among women globally, resulting in considerable psychological and physical distress for patients. Our previous study synthesized a novel derivative, 2-Deoxy-Rh2, which exhibited anticancer properties by influencing glycolysis and mitochondrial respiration. The objective of the current study was to investigate the anti-proliferative effects and underlying mechanisms of 2-Deoxy-Rh2 on human breast cancer cell lines MCF-7 and MDA-MB-231. In our experiments, we observed that 2-Deoxy-Rh2 reduced cell viability and induced cell cycle arrest, reactive oxygen species accumulation, and mitochondrial dysfunction. Furthermore, treatment with 2-Deoxy-Rh2 affected autophagic flux and induction, leading to increased expression of microtubule-associated protein light chain 3B (LC3B) and decreased expression of sequestosome 1 (P62) expression in both two breast cancer cell lines, which could be reversed by 3-Methyladenine (3-MA). Additionally, the AMPK signaling pathway plays a crucial role in 2-Deoxy-Rh2-induced autophagy. 2-Deoxy-Rh2 modulated the expression levels of mTOR and AMPK in MCF-7 and MDA-MB-231 cells, resulting in the cellular homeostasis disruption, autophagy and apoptosis, which was further corroborated by compound C (CC). Finally, the study validated the antitumor activity and mechanism of 2-Deoxy-Rh2 in vivo using Balb/c mice bearing 4T1 tumor cells. Overall, the results suggest that 2-Deoxy-Rh2 can induce apoptosis and autophagic cell death through the AMPK/mTOR signaling pathway, positioning it as a promising candidate for an antitumor agent against breast cancer.