Background <p>Neuroinflammation is caused by the overactivation of microglia, contributing to secondary brain injury in ischemic stroke. Oxytocin (OXT), a neuropeptide synthesized by neurons in the paraventricular nucleus (PVN) of the hypothalamus, has demonstrated potential in mitigating inflammatory responses across various pathological conditions. However, research on the role of PVN^OXT neurons in ischemic stroke is limited, and the modulatory effect of these neurons on neuroinflammation remains unclear.</p> Methods <p>Transient middle cerebral artery occlusion (tMCAO) was performed in mice. OXT levels were measured in the peri-infarct cortex, serum, and cerebrospinal fluid (CSF), and OXTR expression was assessed in the peri-infarct cortex after tMCAO. Chemogenetic approaches were used to selectively activate PVN^OXT neurons in OXT-Cre mice, after which neurological function, infarct volume, and blood–brain barrier integrity were evaluated. To investigate the mechanisms underlying the regulation of ischemic injury by PVN^OXT neurons, RNA sequencing of the ipsilateral ischemic hemisphere was performed. Additional analyses, including flow cytometry, molecular assays, and Transwell migration experiments, were conducted to validate the downstream signaling pathways and cellular responses.</p> Results <p>OXT levels significantly reduced in the peri-infarct cortex, serum, and CSF, whereas OXTR expression increased in the peri-infarct cortex after tMCAO. Chemogenetic activation of PVN^OXT neurons increased OXT levels in both the brain and circulation, reduced infarct volume, and improved neurological outcomes. In addition, transcriptomic analysis identified CXCL3 as one of the most significantly downregulated chemokines after the activation of PVN^OXT neurons, and CXCL3 downregulation was associated with reduced neutrophil chemotaxis. Further in vivo and in vitro investigations demonstrated that PVN^OXT neurons inhibit microglial CXCL3 expression via the OXTR–ERK signaling pathway, thereby restricting the infiltration of neutrophils. In contrast, the administration of recombinant CXCL3 promoted the recruitment of neutrophils and exacerbated ischemic injury.</p> Conclusions <p>PVN^OXT neurons alleviate post-ischemic brain injury by inhibiting the secretion of CXCL3 from microglia, consequently reducing neutrophil chemotaxis. These results underscore the therapeutic potential of targeting PVN^OXT neurons and their downstream signaling pathways to mitigate immune-mediated damage in ischemic stroke.</p>