Sirtuin-3 (Sirt3) deacetylates several mitochondrial proteins implicated into cerebral ischemia/reperfusion (I/R) injury. The mitochondrial unfolded protein response (UPR) favors mitochondrial proteostasis during various stressors. Here, we used Sirt3 transgenic mice and a transient middle cerebral artery occlusion model to evaluate the molecular basis of Sirt3 on the UPR during brain post-ischemic dysfunction. The present study illustrated that Sirt3 abundance was suppressed in the brain after brain ischemic abnormalities. Overexpression of Sirt3 suppressed the infarction size and attenuated neuroinflammation after brain I/R injury. Sirt3 overexpression restored neural viability by reducing mitochondrial ROS synthesis, maintaining the mitochondrial potential and improving mitochondrial adenosine triphosphate synthesis. Sirt3 overexpression protected neuronal mitochondria against brain post-ischemic malfunction via eliciting the UPR by the forkhead box O3 (Foxo3)/sphingosine kinase 1 (Sphk1) pathway. Inhibiting either the UPR or the Foxo3/Sphk1 pathway relieved the favorable influence of Sirt3 on neural function and mitochondrial behavior. In contrast, Sphk1 overexpression was sufficient to reduce the infarction size, attenuate neuroinflammation, sustain neuronal viability and prevent mitochondrial abnormalities during brain post-ischemia dysfunction. Thus, the UPR protects neural viability and mitochondrial homeostasis, and the Sirt3/Foxo3/Sphk1 pathway is a promosing therapeutic candidate for ischemic stroke.