Due to sudden loss of cerebral blood circulation, acute ischemic stroke (IS) causes neuronal energy attenuation or even exhaustion by mitochondrial dysfunction resulting in aggravation of neurological injury. In this study, we investigated if Notoginsenoside R1 ameliorated cerebral energy metabolism by limiting neuronal mitochondrial dysfunction in acute IS. Male Sprague–Dawley rats (260–280 g) were selected and performed by permanent middle cerebral artery occlusion model. In vitro, the oxygen glucose deprivation (OGD) model of Neuro2a (N2a) cells was established. We found Notoginsenoside R1 treatment reduced rats' cerebral infarct volume and neurological deficits, with increased Adenosine triphosphate (ATP) level together with upregulated expression of glucose transporter 1/3, monocarboxylate transporter 1 and citrate synthase in brain peri-ischemic tissue. In vitro, OGD-induced N2a cell death was inhibited, cell mitochondrial morphology was improved. Mitochondrial amount, mitochondrial membrane potential, and mitochondrial DNA copy number were increased by Notoginsenoside R1 administration. Furthermore, mitochondrial energy metabolism-related mRNA array found Atp12a and Atp6v1g3 gene expression were upregulated more than twofold, which were also verified in rat ischemic tissue by quantitative polymerase chain reaction (qPCR) assay. Therefore, Notoginsenoside R1 administration increases cerebral glucose and lactate transportation and ATP levels, ameliorates neuronal mitochondrial function after IS. Notoginsenoside R1 may be a novel protective agent for neuronal mitochondria poststroke.