Background <p>Aflatoxin B₁ (AFB₁), a potent mycotoxin, commonly contaminates feeds like maize and soybean, jeopardizing animal reproduction. Although AFB₁ exposure is known to cause oxidative stress, immune activation, and cell death in oocytes and early embryos of several species, its effects on sheep remain unclear. This study aimed to investigate AFB₁-induced damage in ovine oocytes and its underlying mechanisms. Quercetin (QT), a cost-effective flavonoid with antioxidant and anti-inflammatory properties, is potential to improve this damage. The mechanisms may involve non-canonical ferroptosis, an iron-dependent, lipid peroxidation-driven cell death pathway independent of canonical regulators GPX4 and TFR1.</p> Methods <p>4D Fast DIA-based micro-scale quantitative proteomics was conducted to identify the target proteins, and then immunofluorescence, qPCR, and parallel reaction monitoring (PRM) were conducted for the expression validation of the target proteins, and a series of analyses combined with the inhibitor experiments were conducted for the functional validation of the target proteins, including assessments of mitochondrial function (membrane potential ΔΨm, distribution, ATP levels), mitochondrial morphology observation by transmission electron microscopy (TEM), lipid peroxidation detection (LPO imaging, ROS and GSH detection), Fe²⁺ detection, endoplasmic reticulum staining, and early apoptosis signaling detection.</p> Results <p>OPA1 and ACSL4 are screened as the target proteins by micro-scale quantitative proteomics analysis, and immunofluorescence, qPCR and PRM validate their expression. Molecular docking reveals that there is an interaction of OPA1 and ACSL4, and QT exhibits stronger binding affinity to both OPA1 and ACSL4 than AFB₁. AFB₁ induces aberrant upregulation of OPA1 and ACSL4, disrupting mitochondrial cristae and membrane structure, impairing mitochondrial function and energy metabolism (including decreased mitochondrial membrane potential, abnormal distribution, and reduced ATP synthesis), promoting lipid peroxidation and Fe²⁺ accumulation, exacerbating endoplasmic reticulum stress and altering ROS/GSH levels, ultimately leading to ferroptosis in oocytes. Addition of QT ameliorates the AFB₁-induced abnormal expression of OPA1 and ACSL4.</p> Conclusions <p>QT alleviates AFB₁-induced damage on ovine oocytes by suppressing non-canonical ferroptosis via the OPA1/ACSL4 pathway. These findings elucidate a novel mechanism underlying AFB₁-mediated reproductive toxicity and provide a theoretical foundation for applying QT to mitigate AFB₁-induced reproductive impairment and improve livestock health.</p> Graphical Abstract <p></p>