BACKGROUND: Early screening is critical for the prevention of ischemic stroke. miR-574-5p was considered a promising biomarker for ischemic stroke but lacks direct confirmation. This study evaluated miR-574-5p in discriminating ischemic stroke and predicting the severity and prognosis of patients, aiming to provide novel insights into the clinical prevention of ischemic stroke. METHODS: The clinical significance of miR-574-5p was evaluated in 103 ischemic stroke patients with 87 healthy individuals as control. The potential of serum miR-574-5p in the diagnosis and prognosis of ischemic stroke was assessed by ROC and logistic regression analyses. In vitro, oxygen-glucose deprivation (OGD)-induced microglia was established. The regulation of inflammation, oxidative stress, and proliferation of microglia by miR-574-5p were assessed by cell transfection. The downstream targets of miR-574-5p were predicted from public databases, and the targeting relationship was evaluated by luciferase reporter assay. RESULTS: Reducing serum miR-574-5p was observed in ischemic stroke patients relative to healthy individuals, which discriminated ischemic stroke patients. Serum miR-574-5p was negatively correlated with the NIHSS score of ischemic stroke patients and was identified as a risk factor for patients' adverse prognosis. In OGD-induced microglia, overexpressing miR-574-5p could alleviate OGD-induced inflammation and oxidative stress and promote cell growth. Among predicted targets, ATP2B2 was upregulated in ischemic stroke and showed a negative correlation with miR-574-5p. miR-574-5p negatively regulated ATP2B2 in OGD-induced microglia, and the overexpression of ATP2B2 reversed the protective effect of miR-574-5p. CONCLUSION: miR-574-5p acted as a biomarker for ischemic stroke and mediated neuroinflammation via targeting ATP2B2.