All pathogenic flaviviruses examined thus far inhibit host interferon (IFN) responses by suppressing the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Both Langat virus (LGTV; a member of the tick-borne encephalitis virus serogroup) and Japanese encephalitis virus use the nonstructural protein NS5 to suppress JAK-STAT signaling. However, NS5 is also critical to virus replication, contributing methyltransferase and RNA-dependent RNA polymerase (RdRP) activities. The specific amino acid residues of NS5 involved in IFN antagonism are not known. Here, we demonstrate that the LGTV NS5 JAK-STAT inhibitory domain is contained between amino acids 355 and 735 (of 903), a range which lies within the RdRP domain. Furthermore, we identified two noncontiguous stretches of specific amino acids within the RdRP, 374 to 380 and 624 to 647, as critical for inhibition of JAK-STAT signaling. Despite considerable separation on the linear NS5 sequence, these residues localized adjacent to each other when modeled on the West Nile virus RdRP crystal structure. Due to the general conservation of RdRP structures, these results suggest that the specific residues identified act cooperatively to form a unique functional site on the RdRP responsible for JAK-STAT inhibition. This insight into the mechanism underlying flavivirus IFN evasion strategies will facilitate the design of antiviral therapeutics that potentiate the action of IFN during infection.