Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that is the most significant etiological agent of childhood viral neurological infections. However, no specific antiviral drug is currently available to treat JEV infections. The JEV envelope (E) protein is a class II viral fusion protein that mediates host cell entry, making interference with the interaction between the E protein of JEV and its cognate receptors an attractive strategy for anti-JEV drug development. In this study, we identified a peptide derived from a phage display peptide library against the E protein of JEV, designated P1, that potentially inhibits in vitro and in vivo JEV infections. P1 inhibits JEV infection in BHK-21 cells with 50% inhibitory capacity at a concentration of 35.9 μM. The time-of-addition assay indicates that JEV replication is significantly inhibited during pre-infection and co-infection of P1 with JEV while post-infection treatments with P1 have very little impact on JEV proliferation, showing that P1 inhibits JEV infection at early stages and indicating the potential prophylactic effect of P1. We adapted an in vitro BiFC assay system and demonstrated that P1 interacts with JEV E proteins and blocks their entry into cells. We also evaluated the therapeutic efficacy of P1 in a lethal JEV mouse model exhibiting systemic and brain infections. Interestingly, P1 treatment protected C57BL/6 mice against mortality, markedly reduced the viral loads in blood and brain, and diminished the histopathological lesions in the brain cells. In addition to controlling systemic infection, P1 has a very low level of cytotoxicity and acts in a sequence-specific manner, as scrambled peptide sP1 does not show any antiviral activity. In conclusion, our in vitro and in vivo experimental findings show that P1 possesses antiviral activity against JEV infections, is safe to use, and has potential for further development as an antiviral treatment against JEV infections. •Peptide P1 target to E protein by phage display screening inhibits JEV infection by blocking JEV attachment to host cells.•P1 decreases histopathological damages and viral load in mouse brain.•P1 increases survival rate of mice in lethal JEV challenge after preincubation with JEV.