MicroRNA-22 (miR-22) has been reported to exert a neuroprotective effect. However, the specific role and mechanism of miR-22 in ischemia/reperfusion (I/R)-induced brain injury are still not known well. In this study, we evaluated whether miR-22 participates in I/R-induced neuronal injury and the potential mechanism by using an oxygen-glucose deprivation/reperfusion (OGD/R) model in vitro. Our results showed that miR-22 was significantly down-regulated in SH-SY5Y cells suffering from OGD/R. Up-regulation of miR-22 by its specific mimic could protect SH-SY5Y cells against OGD/R-induced injury. The luciferase reporter assay demonstrated that T-cell lymphoma invasion and metastasis 1 (Tiam1) was a direct target of miR-22. MiR-22 mimic obviously inhibited Tiam1 expression in OGD/R-exposed SH-SY5Y cells. Tiam1 siRNA could attenuate OGD/R-induced SH-SY5Y cell injury. In addition, Tiam1 siRNA reduced the activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) in OGD/R-exposed SH-SY5Y cells, and up-regulation of Rac1 activity could attenuate the neuroprotective effect of miR-22 up-regulation. Furthermore, OGD/R exposure led to increased methylation of miR-22, and the demethylating agent 5-Aza-dC significantly up-regulated miR-22 expression and inhibited Tiam1 expression and Rac1 activation. Taken together, our results demonstrated that DNA methylation-mediated miR-22 down-regulation aggravated I/R-induced neuron injury by promoting the activation of Tiam1/Rac1 signals. Our findings provide a deeper understanding of I/R-induced brain injury and suggest that miR-22 may be a promising therapeutic target for this disease.