Very little information is available on the role of innate immune mechanisms in Japanese encephalitis virus (JEV) infection. This study was designed to investigate the role of all Pattern Recognition Receptors (PRRs) in JEV infection in a mouse neuronal cell line in comparison to events that occur in vivo, using JEV infected suckling and adult mice. Analysis of mRNA expression was carried out using RT-PCR for detection of PRR genes and their downstream pathway genes, while a PCR array technique was used to examine the complete transcription analysis. Amongst the various innate immune receptors, TLR3 gene exhibited differential expression in JEV-infected Neuro2a, in suckling mice and adult mouse brain cells but not in uninfected control cells. The downstream events of TLR3 were confirmed by increased mRNA expression of IRF3 and interferon-β in JEV-infected Neuro2a cells and suckling mice brain tissue. To confirm the functional significance of this observation, TLR3 gene silencing experiments were carried using specific siRNA in Neuro2a cells. The results revealed a significant enhancement of JEV replication in TLR3 gene silenced JEV-infected Neuro2a cells, thereby suggesting that TLR3 serves a protective role against JEV. The expression levels of other PRRs varied. JEV-infected adult mice showed significant upregulation of TLR2 and MDA5 as compared to JEV-infected suckling mice and Neuro2a cells. In addition, upregulation of Myd88 and IRF7 was also noted in adult mice. These observations, coupled with the fact that adult mice infected with JEV exhibited longer survival rates, suggests that the host antiviral TLR2 response seen in adult mice was eventually countered by the virus by using MDA5 receptor. Our findings suggest that different PRRs appear to be involved in JEV infection in Neuro2a cells and brains of suckling and adult mice.