In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenicity of both DNA and RNA viruses. Previously, we reported the successful application of this strategy to control the neurovirulent phenotype of a model chimeric tick-borne encephalitis/dengue type 4 virus (TBEV/DEN4), containing the structural protein genes of a highly virulent TBEV in the genetic backbone of non-neuroinvasive DEN4 virus. In the present study, we investigated the suitability of this approach for the attenuation of the more neurovirulent chimeric virus (TBEV/LGTV), which is based on the genetic backbone of the naturally attenuated member of the TBEV serocomplex, a Langat virus (LGTV). Unlike the TBEV/DEN4, the TBEV/LGTV virus retained the ability of its parental viruses to spread from the peripheral site of inoculation to the CNS. We evaluated ten potential sites in the 3′NCR of the TBEV/LGTV genome for placement of microRNA (miRNA) targets and found that the TBEV/LGTV genome is more restrictive for such genetic manipulations compared to TBEV/DEN4. In addition, unlike TBEV/DEN4 virus, the introduction of multiple miRNA targets into either the 3′NCR or ORF of the TBEV/LGTV genome had only a modest effect on virus attenuation in the developing CNS of highly permissive newborn mice. However, simultaneous miRNA-targeting in the ORF and 3′NCR had synergistic effect on control and silencing of virus replication in the brain and completely abolished the virus neurotropism. Furthermore, neuroinvasiveness of miRNA-targeted TBEV/LGTV viruses in very sensitive immunodeficient SCID mice was significantly limited. Immunocompetent animals immunized with such viruses were completely protected against challenge with the neurovirulent LGTV parent. These findings support the rationale of the miRNA-targeting approach to develop live attenuated virus vaccines against various neurotropic viruses.