Background <p>8-Chloro-adenosine (8-Cl-Ado) is a promising antitumor agent, and ferroptosis plays a critical role in breast cancer progression. Our previous work demonstrated that 8-Cl-Ado inhibits breast cancer cell proliferation by targeting adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme. However, whether 8-Cl-Ado exerts its anti-tumor effects through the modulation of ferroptosis remains largely unknown.</p> Methods <p>The effects of 8-Cl-Ado on ferroptosis were assessed in vitro and in vivo. The molecular mechanisms of 8-Cl-Ado were investigated by performing bioinformatics analysis, RNA immunoprecipitation assay (RIP), luciferase reporter assay, fluorescence in situ hybridization (FISH), qRT-PCR, and western blotting.</p> Results <p>8-Cl-Ado significantly inhibited the proliferation, migration, and invasion of MCF-7 and MDA-MB-231 breast cancer cells, while promoting ferroptosis, as evidenced by elevated levels of intracellular Fe²⁺, reactive oxygen species (ROS), and malondialdehyde (MDA), along with decreased glutathione (GSH) levels and reduced protein expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). In an orthotopic breast cancer mouse model, 8-Cl-Ado suppressed tumor growth and decreased the expression of SLC7A11 and GPX4. Mechanistically, 8-Cl-Ado downregulated ADAR1 expression, resulting in upregulation of miR-101-3p, which directly targets the 3′UTR of SLC7A11 mRNA, leading to its degradation and subsequent induction of ferroptosis. Moreover, ADAR1 bound to the precursor of miR-101-3p and impairs its processing into mature miR-101-3p in an RNA editing-independent manner.</p> Conclusion <p>Our study identifies a novel pathway by which 8-Cl-Ado promotes ferroptosis through the ADAR1/miR-101-3p/SLC7A11 axis to suppress breast cancer progression. These findings highlight the therapeutic potential of 8-Cl-Ado as a ferroptosis-inducing agent by targeting ADAR1.</p>