Ischemic stroke is a common cause of morbidity and mortality worldwide. The current treatment fails to achieve satisfactory results, because interventional therapy as first-line treatment management has a strict time window. In recent years, a large number of studies have confirmed that adenosine, as an inhibitory neurotransmitter, has a protective effect on cerebral ischemic injury. Nevertheless, direct administration of adenosine has many side effects. Previous studies showed that adenosine exerted neuroprotective effects mainly through adenosine receptor A1 (A1 receptor). Therefore, further study on the mechanism of A 1 receptor induced neuroprotection may find new targets for stroke treament. Mitochondrial biogenesis (MB) is a therapeutic target for ischemic stroke, and the nuclear-encoded peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) is a major regulator of MB. However, the influence of A1 receptor on MB and PGC-1α is unclear. In this study, using the middle cerebral artery occlusion (MCAO) model of mice, we evaluated the temporal and spatial effects of A1 receptor after ischemic stroke and verified the neuroprotection of A1 receptor. Neurological scores were used to assess functional changes in mice. At the same time, we observed the effect of activating A1 receptor on MB and PGC-1α, and the effect of knockdown PGC-1α on A1 receptor induced MB in vitro. WB and immunofluorescence were used to detect relevant indicators of MB. In addition, we downregulated PGC-1α in vivo to observe the effects on A1 receptor induced MB and neuroprotection. The findings indicated that A1 receptor was increased and mainly expressed on neurons in the penumbra, further activated A1 receptor after stroke had neuroprotection. In vitro, activation of A1 promotes MB and increases the expression level of PGC-1α, while downregulation of PGC-1α partially reverses the effect of A1 receptor after OGD/R. Down regulation of PGC-1α in the penumbra neurons can reverse the effects of activation of A1 receptor on MB and neuroprotection. Taken together, these findings indicated that A1receptor promotes MB and improves neurological function after ischemic stroke via PGC-1α.