We aimed to investigate the neuroprotective effect of rutin on retinal ganglion cells (RGCs) under ischemia-reperfusion (I/R) conditions and the underlying mechanisms involving microglia polarization and JAK/STAT3 signaling. RGCs isolated from C57/Bl6 mice were co-cultured with BV2 microglial cells under normal or in vitro oxygen-glucose deprivation and reoxygenation (OGD/R) conditions. Rutin's effects were evaluated by assessing cell viability, apoptosis rates, cytokine levels, microglial polarization markers and JAK/STAT3 phosphorylation levels. The specific target is confirmed through the inhibitory effect of rutin on the respectively activated signaling factors. Furthermore, molecular docking analyses elucidated rutin-JAK1 interactions. OGD/R conditions significantly reduced RGC viability, exacerbated by BV2 co-culture. However, both 1 μM and 5 μM rutin treatment dose-dependently enhanced RGC viability, reduced apoptosis, and suppressed pro-inflammatory cytokine levels. Western blot analysis indicated that rutin promoted the M2 microglial phenotype and suppressed JAK/STAT3 signaling. Notably, rutin selectively inhibited JAK1 phosphorylation without affecting STAT3. Molecular docking highlighted potential interaction sites between rutin and specific JAK1 pseudokinase domain. Rutin exerts neuroprotective effects against retinal I/R injury by promoting M2 microglial polarization, potentially through the selective inhibition of JAK1 phosphorylation within the JAK/STAT3 signaling pathway. These findings provide a foundation for the therapeutic potential of rutin in retinal I/R injuries.