The envelope protein domain III (ED3) of West Nile virus is the major virus-specific neutralization domain and harbors most of the critical mutations that induce resistance against antibody-mediated neutralization. We investigated the molecular mechanisms of neutralization resistance by studying the biophysical perturbations of monoclonal antibody (mAb)-resistant mutations on ED3 wild type. Our results showed that although the solution structure between ED3 wild type and mutants was preserved, the mutations that confer the highest degree of resistance to mAbs showed low protein stability and high local dynamic motions. Interestingly, the latter was observed in regions outside the mutation sites, indicating long range communications within ED3. Thus, we hypothesized that the mechanisms involved in resistance to mAb neutralization may include, in addition to mutations in the epitope, long range effects among distant structural elements. This hypothesis is consistent with reported mutations in other flaviviruses whose surfaces are not exposed for the interaction with other macromolecules, yet they confer mAb neutralization resistance.