T helper cell 1 (Th1)-skewed neurotoxicity contributes to the poor outcome of stroke in rodents. Here we elucidate the mechanism of Th1/Th2 shift in acute ischemic stroke (AIS) patients at hyperacute phase, and seek a miRNA-based therapeutic target. MiR-494 levels in blood from AIS patients and controls were detected by real-time PCR. C57BL/6J mice were subjected to transient middle cerebral artery occlusion and cortical neurons were subjected to oxygen-glucose deprivation. Luciferase reporter system, Chromatin immunoprecipitation sequencing (ChIP-Seq), and ChIP polymerase chain reaction (ChIP-PCR) were performed to unveil the possible mechanism. Here, we demonstrated a Th1/Th2 shift occurred and histone deacetylase 2 (HDAC2) was markedly downregulated in lymphocytes of AIS patients. ChIP-seq revealed that HDAC2 binding sites were enriched in regulation of Th1 cytokine production, and ChIP-PCR confirmed that HDAC2 binding was changed at the intron of signal transducer and activator of transcription 4 (STAT4) and the promoter of T-box transcription factor 21 (T-bet) in lymphocyte of AIS patients. MiR-494 was the most significant increased miRNA in lymphocytes of AIS patients, and miR-494-3p directly targeted HDAC2. A strong association existed between miR-494 and Th1 cytokine, and neurological deficit measured by national institute of health stroke scale (NIHSS) of AIS patients. In vitro and in vivo experiments depicted that antagomir-494 reduced Th1 shift-mediated neuronal and sensorimotor functional damage in ischemic stroke mouse in a HDAC2-STAT4 axis-dependent way. We demonstrated that miR-494 inhibition prevented Th1-skewed neurotoxicity through regulation of HDAC2-STAT4 cascade. This article is protected by copyright. All rights reserved.