Ischemic stroke is a common disease with high morbidity and mortality. Remote ischemic preconditioning (RIPC) can stimulate endogenous protection mechanisms by inducing ischemic tolerance to reduce subsequent damage caused by severe or fatal ischemia to non-ischemic organs. This study was designed to assess the therapeutic properties of RIPC in ischemic stroke and to elucidate their underlying mechanisms. Neurobehavioral function was evaluated with the modified neurological severity score (mNSS) test and gait analysis. PET/CT was used to detect the ischemic volume and level of glucose metabolism. The protein levels of cytochrome c oxidase-IV (COX-IV) and heat shock protein 60 (HSP60) were tested by Western blotting. TUNEL and immunofluorescence staining were used to analyze apoptosis and to observe the nuclear translocation and colocalization of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in apoptotic cells. Transmission electron microscopy (TEM) was used to detect mitochondrial-derived vesicle (MDV) production and to assess mitochondrial ultrastructure. The experimental results showed that RIPC exerted significant neuroprotective effects, as indicated by improvements in neurological dysfunction, reductions in ischemic volume, increases in glucose metabolism, inhibition of apoptosis, decreased nuclear translocation of AIF and EndoG from mitochondria and improved MDV formation. In conclusion, RIPC alleviates ischemia/reperfusion injury after ischemic stroke by inhibiting apoptosis via the endogenous mitochondrial pathway.