Here, we explored a possible mechanism of microRNA-126-3p (miR-126-3p) on neonatal rats with hypoxia-reoxygenation injury (HI). After administering HI to newborn Sprague-Dawley rats, the expression of miR-126-3p in the brain injury was assessed by RT-PCR. A miR-126-3p mimic and inhibitor were treated in the HI neonatal rats. The water maze test, TTC, HE, Nissl and TUNEL staining were separately implemented to test the effects of miR-126-3p on the HI-treated neonatal rats. At the same time, the phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) expression in the damaged cortex region was analyzed. In vitro, cortical neurons were cultured and treated with oxygen and glucose deprivation (OGD), then transfected miR-126-3p mimic, PIK3R2 overexpression lentivirus vector or silence of PIK3R2. The cell viability was observed by CCK-8. The autophagy of neurons was detected by acridine orange staining. In contrast to the sham-operated rats, the miR-126-3p expression significantly decreased, but PIK3R2 expression markedly rose in the cortex of HI rats. Injection of miR-126-3p mimic raised the learning and memory abilities through down-regulating the cerebral ischemic area, improving pathological damage of the cortex, reducing the neurons apoptosis of the cortex and down-regulating the autophagy-related and apoptosis-related proteins. Overexpression of PIK3R2, a miR-126-3p target, may reduce cell viability and boost autophagy and apoptosis. Silence of PIK3R2 promoted cell viability and inhibited cell apoptosis and autophagy. The consequences of miR-126-3p were comparable to those of PIK3R2 silencing. A new therapeutic target for HI injury in newborn rats is provided by the overexpression of miR-126-3p, which inhibits autophagy and death of cortical neurons by targeting PIK3R2 in HI-treated neonatal rats.