Growing evidence have suggested that mitophagy could exert a neuroprotective role in brain ischemia by removing the damaged mitochondria. However the upstream mechanisms of mitophagy are remain unclear. We previously observed a decrease of miR-330 in a miRNA profile of plasma from patients within 3 h after a stroke. Our study further focused on the role and mechanism of miR-330 in mitophagy induced by hypoxia-ischemia (H/I) in rats. Cerebral ischemia model in rats was made with permanent middle cerebral artery occlusion (pMCAO). In vitro, ischemic model in primary neurons was established with oxygen-glucose deprivation. Various methods, including TTC staining, immunofluorescence staining, Western blot, ELISA, flow cytometry, and transmission electron microscopy were used to clarify the role of miR-330 after H/I, and whether miR-330/phosphoglycerate mutase family member 5 (PGAM5) axis could regulate dynamin-related protein 1 (Drp-1) mediated mitophagy. MiR-330 levels decreased both in rat plasma and in ipsilateral brain tissues after H/I. Pretreating animals with miR-330 antagomir could decrease cerebral infarction, edema, mortality, and apoptosis after 6-h pMCAO. PGAM5 was validated as a target of miR-330. MiR-330 agomir and antagomir transfection respectively decreased and increased the PGAM5 protein expression. MiR-330 could down-regulate mitophagy by inhibiting PGAM5-induced Drp1 dephosphorylation, thus reducing the recruitment of Drp1 to mitochondrial outer membrane and Drp1-mediated mitophagy after H/I. Our results suggest a role of miR-330 in regulating mitophagy. Our study suggested a novel miR-based intervention strategy for stroke. Copyright © 2020. Published by Elsevier B.V.