Objective: To explore the effect of the K283R mutation in JE/Zika chimeric virus envelope protein on neurovirulence in mice. Methods: Using the full-length cDNA clone of JE/ZIKV (MR766) as the template, the infectious clone carrying E-K283R substitution was constructed via overlap extension PCR-mediated site-directed mutagenesis and molecular cloning, followed by restriction enzyme (XhoⅠ) linearization and in vitro transcription. The mutant virus JE/ZIKV (MR766) (K283R) was rescued by electroporation into BHK21 cells. Viral characteristics were validated through serial passage, sequencing, plaque assay, growth kinetics, and indirect immunofluorescence. Neurovirulence was quantitatively assessed by intracranial challenge in 3-week-old female Kunming mice, with LD50 as the endpoint metric. Results: Restriction analysis confirmed successful construction of the JE/ZIKV (MR766) (K283R) infectious clone. Plaque assays, sequencing, and immunofluorescence verified viral rescue, showed larger plaque diameters (0.20 ± 0.04) cm for the mutant compared to the parental strain JE/ZIKV (MR766) (0.14 ± 0.03) cm, P < 0.05. The growth curve showed that both strains reached peak titers at 60 h post-infection. Animal experiments revealed an intracranial LD50 of 1.35 pfu/0.03 mL for JE/ZIKV (K283R), slightly lower than the parental strain (2.21 pfu/0.03 mL). Conclusion: The K283R substitution in ZIKV E protein did not significantly attenuate murine neurovirulence, suggesting its non-critical role in viral pathogenicity regulation.