The mitochondrial calcium uniporter (MCU) maintains intracellular Ca2+ homeostasis by transporting Ca2+ from the cell cytosol into the mitochondrial matrix and is important for shaping Ca2+ signals and the activation of programmed cell death. Inhibition of MCU by ruthenium red (RR) or Ru360 has previously been reported to protect against neuronal death. The aim of the present study was to analyze the mechanisms underlying the effects of MCU activity in a rat model of cerebral ischemia/reperfusion (I/R) injury. Adult male Wistar rats were divided into 4 groups; sham, I/R, I/R + RR and I/R + spermine (Sper) and were subjected to reversible middle cerebral artery occlusion for 2 h followed by 24 h of reperfusion. A bolus injection of RR administered 30 min prior to ischemia was found to significantly decrease the total infarct volume and reduce neuronal damage and cell apoptosis compared with ischemia/reperfusion values. However, treatment with Sper, an activator of the MCU, increased the injury induced by I/R. Analysis of energy metabolism revealed that I/R induced progressive inhibition of complexes I‑IV of the electron transport chain, decreased ATP production, dissipated the mitochondrial membrane potential and increased the generation of reactive oxygen species. Treatment with RR ameliorated the condition, while spermine had the opposite effect. In conclusion, blocking MCU was demonstrated to exert protective effects against I/R injury and this process may be mediated by the prevention of energy failure.