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Previous studies revealed that Japanese encephalitis virus (JEV) infection alters the expression of miRNA in central nervous system (CNS). However, the mechanism of JEV infection contributes to the regulation of miRNAs in CNS remain obscure. Here, we found that a global degradation of mature miRNA in mouse brain and neuroblastoma cells after JEV infection. In additional, the integrative analysis of miRNAs and mRNAs suggests that those down-regulated miRNAs are primarily targeted inflammation genes and the miR-466d-3p target the IL-1β which in the middle of those inflammation genes. Transfection of miR-466d-3p decreased the IL-1β expression and inhibited the JEV replication in NA cells. Interestingly, the miR-466d-3p level increased after JEV infection in the presence of cycloheximide, which indicated that viral protein expression reduces miR-466d-3p. Therefore, we generated all the JEV coding protein and demonstrated that NS3 is a potent miRNA suppressor. Furthermore, the NS3 of ZIKA virus, WNV, DENV1 and DENV2 also decreased the expression of miR-466d-3p. The in vitro unwinding assay demonstrated that the NS3 could unwind the pre-miR-466d and induce the disfunction of miRNA. Using computational models and RNA immunoprecipitation assay, we found that arginine-rich domains of NS3 are critical for pre-miRNA binding and the degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues revealed that R226G and R202W significantly reduced the binding affinity and degradation of pre-miR-466d. Together, these results extend the helicase of Flavivirus function beyond unwinding duplex RNA to the decay of pre-miRNAs, which provides a new mechanism of NS3 in regulating miRNA pathways and promoting the neuroinflammation.Author SummaryHost miRNAs had been reported to regulate JEV induced inflammation in central nervous system. We found that the NS3 of JEV can reduce most of host miRNA expression. The helicase region of the NS3 specifically binds to precursors of miRNA and lead to incorrect unwinding of precursors of miRNAs which inhibits the function of miRNAs. This observation leads to two major findings. First, we identified the miR-466d-3p targets to the host IL-1β and E protein of JEV, and NS3 degrades the miR-466d-3p to promote the brain inflammation and viral replication. Second, we proved that the arginine on the helicase of NS3 is the main miRNA binding sites, and the miRNA degradation by NS3 was abolished when the R226 and R202 were mutated on the NS3. These findings were also confirmed with NS3 of ZIKA virus, WNV and DENV which could decrease the expression level of miR-466d-3p to enhance the inflammation. Our study provides new insights into the molecular mechanism of encephalitis caused by JEV, and reveals several amino acid sites to further attenuate the JEV vaccine.