The role of PD-L1/PD-1 axis in IS remains controversial, with conflicting outcomes from systemic PD-1 or PD-L1 knockout models. These discrepancies underscore the complexity of PD-L1/PD-1 signaling and highlight the need to explore its cell-specific functions, particularly in neurons and microglia. Neurons, as the primary functional cells in the CNS, play a critical role in modulating local immune responses, yet their contribution to PD-L1/PD-1 signaling in IS is unknown. Furthermore, the mechanisms linking PD-L1/PD-1 to microglial polarization remain unclear. This study investigates whether targeted suppression of neuronal PD-L1 alleviates IS injury by modulating the PD-1/RFX1 axis and driving microglial polarization toward an anti-inflammatory phenotype.Middle cerebral artery occlusion (MCAO) was performed in mice to model IS. Neuronal PD-L1 was selectively suppressed using AAV. Microglial polarization, PD-1 and RFX1 expression, and neuroinflammation were assessed via flow cytometry, immunofluorescence, 4D-FastDIA proteomics, and qPCR/ELISA. In vitro, PD-1 knockdown BV2 cells and RFX1 overexpression models were established to validate mechanistic interactions.Neuronal PD-L1 suppression reduced infarct volume, improved cerebral blood flow, and alleviated neurological deficits in ischemic stroke mice. Microglial PD-1 expression decreased significantly, accompanied by a phenotypic shift from pro-inflammatory to anti-inflammatory states. RFX1, exclusively expressed in microglia, was downregulated and identified as a key regulator of PD-1 and microglial polarization. In vitro, RFX1 overexpression reversed the anti-inflammatory effects of PD-1 knockdown, restoring pro-inflammatory cytokine levels. Critically, neuronal PD-L1 suppression spared peripheral immune cells, avoiding systemic immune disruption. These findings establish the PD-1/RFX1 axis as a central mediator of neuron-microglia crosstalk in IS neuroinflammation.Neuronal PD-L1 suppression attenuates IS injury by modulating the PD-1/RFX1 axis to promote anti-inflammatory microglial polarization. This study reveals a novel neuron-microglia crosstalk mechanism and highlights RFX1 as a therapeutic target for IS. The neuron-specific strategy overcomes limitations of systemic PD-L1/PD-1 inhibition, offering a precise and clinically translatable approach to mitigate neuroinflammation while preserving peripheral immune homeostasis.Copyright © 2025 The Author(s). Published by Wolters Kluwer Health, Inc.