Sirtuin 6 (SIRT6), a member of the sirtuin family of NAD(+)-dependent deacetylases, has been shown to produce beneficial effects in myocardial ischemia/reperfusion (I/R). However, the role of SIRT6 in cerebral I/R is largely unclear. In this study, we investigated the effects of SIRT6 overexpression in regulating I/R injury in a mouse cerebral I/R model in vivo and in oxygen-glucose-deprivation/reoxygenation (OGD/R)-stimulated neuro-2a neuroblastoma cells in vitro. We found that cerebral I/R (1 h/24 h) resulted in decreased SIRT6 expression in the cerebral cortex (P < 0.01). SIRT6 overexpression in the brain by in vivo gene transfer enhanced the antioxidant NRF2 signaling (P < 0.05), reduced oxidative stress (P < 0.05), and attenuated cerebral I/R-induced brain tissue damage and neurological deficits (P < 0.05). These neuroprotective effects of SIRT6 overexpression were abolished in NRF2 knockout mice. In neuro-2A neuroblastoma cells, SIRT6 overexpression increased total and nuclear NRF2 levels (P < 0.05), reduced oxidative stress (P < 0.05), and attenuated OGD/R-induced cell death (P < 0.05); these protective effects were blocked by NRF2 knockdown (P < 0.05). Moreover, in OGD/R-stimulated neuro-2A cells, SIRT6 overexpression produced similar protective effects to those induced by the antioxidant NAC, but no added benefits were detected when SIRT6 overexpression was used in combination with NAC (P > 0.05). These findings provide evidence that SIRT6 can protect the brain from cerebral I/R injury by suppressing oxidative stress via NRF2 activation. Thus, SIRT6 may serve as a potential therapeutic target for ischemic stroke.