<p>Cisplatin resistance continues to pose a significant challenge in the treatment of osteosarcoma, markedly impairing clinical outcomes. Accumulating evidence indicates that exosomal microRNAs (miRNAs) facilitate intercellular communication and play a critical role in drug resistance mechanisms. In this study, we conducted high-throughput sequencing of serum-derived exosomes and identified miR-20a-5p as significantly upregulated in cisplatin-resistant osteosarcoma patients. Functional analyses revealed that exosomes enriched with miR-20a-5p derived from cisplatin-resistant HOS cells (HOS-DDP) were internalized by sensitive HOS cells, thereby promoting cisplatin resistance through the inhibition of ferroptosis. Mechanistically, co-culture with miR-20a-5p - rich exosomes resulted in preserved mitochondrial morphology, reduced Fe²⁺ and ROS accumulation, increased GSH levels, and suppressed T-SOD depletion under cisplatin-induced stress. Furthermore, exosomal miR-20a-5p was demonstrated to directly target and suppress the tumor suppressor RUNX3, a key regulator of ferroptotic pathways. Overexpression of RUNX3 restored ferroptosis and reversed the miR-20a-5p–mediated resistance phenotypes. Collectively, these findings elucidated that exosomal miR-20a-5p conferred cisplatin resistance in osteosarcoma by inhibiting ferroptosis via RUNX3 suppression, providing a promising therapeutic target for overcoming chemoresistance.</p>