Background and Purpose— Sirtuin 1 (SIRT1) is a member of NAD ⁺ -dependent protein deacetylases implicated in a wide range of cellular functions and has beneficial properties in pathologies including ischemia/reperfusion processes and neurodegeneration. However, no direct evidence has been reported on the direct implication of SIRT1 in ischemic stroke. The aim of this study was to establish the role of SIRT1 in stroke using an experimental model in mice. Methods— Wild-type and Sirt1 ^−/− mice were subjected to permanent focal ischemia by permanent ligature. In another set of experiments, wild-type mice were treated intraperitoneally with vehicle, activator 3 (SIRT1 activator, 10 mg/kg), or sirtinol (SIRT1 inhibitor, 10 mg/kg) for 10 minutes, 24 hours, and 40 hours after ischemia. Brains were removed 48 hours after ischemia for determining the infarct volume. Neurological outcome was evaluated using the modified neurological severity score. Results— Exposure to middle cerebral artery occlusion increased SIRT1 expression in neurons of the ipsilesional mouse brain cortex. Treatment of mice with activator 3 reduced infarct volume, whereas sirtinol increased ischemic injury. Sirt1 ^−/− mice displayed larger infarct volumes after ischemia than their wild-type counterparts. In addition, SIRT1 inhibition/deletion was concomitant with increased acetylation of p53 and nuclear factor κB (p65). Conclusions— These results support the idea that SIRT1 plays an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53-induced and nuclear factor κB-induced inflammatory and apoptotic pathways.