<p>This study aimed to elucidate the neuroprotective effects of miR-122 in ischemic brain injury and its unique dual mechanism, simultaneously targeting both neuroinflammation and ferroptosis. Using a rat tMCAO model, we found that agomiR-122 treatment significantly improved neurological severity scores (mNSS) and rotarod performance, alleviated neuronal damage as well as mitochondrial dysfunction (CCO activity, ATP leves), and concurrently reduced serum levels of pro-inflammatory cytokines (TNF-α, IL-1β) and ferroptosis markers (Fe²⁺, MDA). Mechanistically, this study to integrate bioinformatics prediction with dual-luciferase reporter assays to confirm that PI3K is a direct target of miR-122. In vivo, miR-122 overexpression activated the PI3K/Akt pathway, upregulated mRNA expression of SLC7A11 and GPX4, downregulated ACSL4, and suppressed the expression of the microglial marker Iba1 and pro-inflammation M1 marker CD86 while further enhancing the expression of the anti-inflammatory marker CD206. In vitro OGD/R models further demonstrated that the miR-122 mimic could inhibit Iba1 expression, improve mitochondrial morphology, and elevate mitochondrial membrane potential, while also upregulating the expression of p-PI3K/PI3K, p-Akt/Akt, SLC7A11, and GPX4 proteins, and downregulating ACSL4. Notably, these protective effects were reversed by the PI3K inhibitor LY294002. In conclusion, miR-122 confers protection by directly targeting PI3K and activating its downstream Akt pathway, thereby establishing a unified regulatory axis that coordinately suppresses neuroinflammation and ferroptosis.</p>