MiR-128, one of the most enriched miRNAs in the human brain, has been reported to protect MCAO mice via inhibiting P38α MAPK. Whether it is involved in pathogenesis in acute ischemic stroke patients remains to be determined. The present study focused on the clinical importance of miR-128 and its underlying mechanisms. We detected miR-128 levels in the circulating lymphocytes, neutrophils, and plasma of acute ischemic stroke patients by using RT-PCR. miR-128 levels were significantly elevated in circulating lymphocytes, neutrophils, and plasma of patients with acute ischemic stroke. In addition, miR-128 levels in circulating lymphocytes correlated positively with the infarction volume, NIHSS scores at 7 days and mRS at 90 days after ischemic stroke onset. Subsequent KEGG pathway analysis showed that the MAPK signaling pathway and cell cycle are among the pathways targeted by miR-128. Although no correlation was found between miR-128 in plasma and peripheral inflammatory cell numbers, miR-128 decreased in the penumbra and increased in the infarction core of ipsilateral brain tissues in MCAO mice. Moreover, an in vitro study demonstrated that miR-128 antagomir aggravated primary neuronal damage and exacerbated cell cycle reactivation induced by OGD/R stimulation; the underlying mechanism involved increasing cyclin A2, PTEN, and ERK expression and promoting phosphorylation of PTEN and ERK. From the above results, we concluded that the upregulation of miR-128 in circulating lymphocytes of acute ischemic stroke patients was correlated with stroke severity and miR-128 antagomir exacerbated ischemia-reperfusion induced neuronal injury via promoting neuronal cell cycle reentry.