MicroRNAs (miRNAs) have been suggested as pivotal regulators in the pathological process of cerebral ischemia and reperfusion injury. In this study, we aimed to investigate the role of miR-135a in regulating neuronal survival in cerebral ischemia and reperfusion injury using an in vitro cellular model induced by oxygen-glucose deprivation and reoxygenation (OGD/R). Our results showed that miR-135a expression was significantly decreased in neurons with OGD/R treatment. Overexpression of miR-135a significantly alleviated OGD/R-induced cell injury and oxidative stress, whereas inhibition of miR-135a showed the opposite effects. Glycogen synthase kinase-3β (GSK-3β) was identified as a potential target gene of miR-135a. miR-135a was found to inhibit GSK-3β expression, but promote the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and downstream signaling. However, overexpression of GSK-3β significantly reversed miR-135a-induced neuroprotective effect. Overall, our results suggest that miR-135a protects neurons against OGD/R-induced injury through downregulation of GSK-3β and upregulation of Nrf2 signaling.