Our previous study demonstrated that microRNA-424 (miR-424) protected against experimental stroke through inhibition of microglial proliferation and activation by targeting cell cycle proteins. The purpose of this study was to further explore the clinical significance of miR-424 in peripheral immune cells of patients with acute ischemic stroke (AIS). Blood samples were collected from 40 patients within 6 hours of symptom onset and 27 control subjects. MiR-424 levels in lymphocytes, neutrophils and plasma were determined by quantitative realtime-PCR. The diagnostic sensitivity and specificity of miR-424 for stroke was evaluated by receiver operator characteristic (ROC) curve. The correlation between miR-424 levels and clinical data was analyzed using Pearson's correlation test. Plasma levels of inflammatory mediators (TNF-alpha, IL-10) and neurotrophic factor (IGF-1) were detected by ELISA. Notably, miR-424 expression levels in lymphocytes and neutrophils increased after stroke, suggestive of its diagnostic value in ischemic stroke. MiR-424 levels in neutrophils were negatively correlated with infarct volume. Lymphocytic miR-424 levels were negatively correlated with the number of lymphocytes and the expression of cyclin-dependent kinase CDK6. Moreover, plasma TNF-alpha and IGF-1 levels increased and decreased, respectively, in stroke patients, and miR-424 levels in lymphocytes and neutrophils were both inversely correlated with plasma TNF-alpha, IL-10, or IGF-1 levels. In summary, miR-424 levels in peripheral immune cells has diagnostic potential for ischemic stroke, and might affect the severity of acute stroke by depressing the peripheral inflammatory response through CDK6-dependent pathway in lymphocytes or CDK6-independent pathway neutrophils.