Remote ischemic pre-conditioning (RIPC) protects against ischemia/reperfusion (I/R) injury. However, the mechanisms underlying this protection remain unclear. In the present study, we investigated the role of Janus-activated kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway and cell cycle arrest, and their relationship with neuronal apoptosis following RIPC.A rat cerebral I/R injury model was induced by middle cerebral artery occlusion (MCAO), and AG490 was used to investigate the mechanisms of RIPC. p-JAK2-, p-STAT3-, cyclin D1-, and cyclin-dependent kinase 6 (CDK6) expression was assessed by Western blotting and immunofluorescence staining.RIPC reduced the infarct volume, improved neurological function, and increased neuronal survival. Furthermore, p-JAK2 and p-STAT3 were detected during the initial phase of reperfusion; the expression levels were significantly increased at 3 and 24 h after reperfusion and were suppressed by RIPC. Additionally, the MCAO-induced upregulation of the cell cycle regulators cyclin D1 and CDK6 was ameliorated by RIPC. Meanwhile, cyclin D1 and CDK6 were colocalized with p-STAT3 in the ischemic brain.RIPC ameliorates the induction of the JAK2/STAT3 pathway and cell cycle regulators cyclin D1 and CDK6 by MCAO, and this net inhibition of cell cycle re-entry by RIPC is associated with downregulation of STAT3 phosphorylation.© 2022 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.