Japanese encephalitis virus is one of the important CNS related pathogens for which no specific treatment available.Therefore, in the present study, we have developed a hydroalcoholic formulation of belladonna (B200) and elucidated its anti-JEV activity using neuronal and microglia cell culture models. Characterization of B200 by UPLC-PDA showed the presence of 87.76% of atropine and 6.31% of scopolamine. Neuronal cell survival assay showed that7.01μg/ml was the EC 50 of B200 which was further confirmed by reduction in necrotic cell population by and reduction in caspase 3 and 8 enzymatic activities. Antiviral effect of B200 was confirmed by reduction in intracellular JEV level. To decipher the mechanism of antiviral, whole-transcriptome sequencing was performed, and crucial pathways involved in B200 mediated neuroprotection during JEV infection were determined. Gene expression analysis revealed that B200 reducesthe pro-apoptotic gene expression, cytokines and inflammatory gene expression observed by significant reduction in BAD, BAX, CASP3, CASP8, IL1B and CXCL10 expression and increase in IL10 responsive genes. Interestingly, our molecular docking analysis revealed that atropine and scopolamine interact with His288 residue of NS3 protein which is crucial for RNA unwinding and ATPase activity that was further confirmed by degradation of NS3 protein. Drug likeness, ADME and toxicity analysis further suggests that atropine and scopolamine both crosses the blood brain barrier which is crucial for effective treatment of JE. These data suggest that belladonna alkaloids exhibit anti-JEV property and reduce microglia activation and inflammatory responses.