MiR-582-5p has been demonstrated to protect against ischemic stroke. However, its implication in the progression of neonatal hypoxic-ischemic encephalopathy (HIE) has not been explored. In this study, we used an in vitro model of oxygen-glucose deprivation (OGD) to investigate the protective effect of miR-582-5p on PC12 cells. OGD-induced inhibition of cell viability and promotion of cell death was assessed by CCK-8 assay and flow cytometry. Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were utilized to examine the levels of inflammatory cytokines. The effects of miR-582-5p on OGD-induced oxidative injury were assessed by the determination of oxidative stress indicators. Furthermore, dual-luciferase reporter assay and gain-offunction assay were used to determine the mechanism of miR-582-5p in OGD-induced cell injury. The expression of miR-582-5p was reduced upon OGD treatment in PC12 cells. Overexpression of miR-582-5p inhibited OGD-induced PC12 cell injury by regulating cell viability, apoptosis, inflammatory responses, and oxidative stress. MiR-582-5p targeted and negatively regulated high mobility group box 1 (HMGB1). MiR-582-5p presented protective effects on OGD-induced PC12 cell injury by targeting HMGB1. Our results indicated that miR-582-5p ameliorates neuronal injury by inhibiting apoptosis, inflammation, and oxidative stress through targeting HMGB1.