Changes in circulating microRNAs (miRNAs) in the cerebrospinal fluid (CSF) have been associated with different neurological diseases. Here, we presented results of a pilot study aimed at determining the feasibility of detecting miRNAs in the CSF of Japanese Encephalitis virus (JEV) infected individuals with acute encephalitis syndrome (AES). We demonstrated the circulating miRNA profile in CSF of acute encephalitis patients infected with JEV. Using a quantitative real-time PCR-based miRNA array, we examined the level of 87 miRNAs expressed in human exosomes isolated from CSF. Subsequently, correlation between cytokine level and miRNAs expression in CSF samples was examined. In this study, we identified and validated the upregulated expression of three miRNAs, miR-21-5p, miR-150-5p, and miR-342-3p that were specifically circulated in CSF of acute encephalitis patients infected with JEV. CSF miR-21-5p, miR-150-5p, and miR-342-3p expressions were also elevated in infected mice brain. However, the expression pattern of these miRNAs differed in neuronal cells, microglial cells, and the exosome derived from JEV-infected cell culture supernatant. Interestingly, neuronal cells infected with vaccine strain (SA-14-14) did not lead to any upregulation of these three miRNAs. Further, miR-150-5p expression was found to be negatively correlated(r = −0.5279, p = 0.016) with TNFα level. Pathway analysis of putative target genes of these miRNAs indicated involvement of TGF-β, NGF, axon guidance, and MAPK signaling pathways in JEV/AES patients. This study for the first time represents the circulating miRNA in CSF of AES patients and identified the upregulated miRNAs in JEV-infected patients and offers the basis for future investigation.