Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen in the family Flaviviridae and causes one of the most severe human neuroinfections. In this study, a neutralizing mouse mAb 14D5, which was previously shown to have cross-reactive binding to several flaviviruses belonging to the TBEV group, was examined for its prophylactic and therapeutic effects in BALB/c mice infected with TBEV. Before and after infection, mice were administrated mAb 14D5 at doses 100 μg and 10 μg per mouse. mAb 14D5 showed clear protective efficacy when injected at the high dose one day after infection, with survival rates that were TBEV dose-dependent. Prophylactic administration of mAb 14D5 was more effective than post-exposure administration and complete protection was documented when the mAb was administered one day before infection. The protective efficacy of mAb 14D5 was significantly higher than that of the anti-TBE serum immunoglobulin. However, no protection was observed in mice received the low dose of mAb 14D5 independent of the timing of mAb injection and TBEV dose. The ability of species-matched mAb 14D5 to mediate TBEV infection in mice was also investigated, and the results indicated that mAb 14D5 did not augment TBEV infection independent of the time of mAb administration. The neutralizing epitope for mAb 14D5 was localized in domain III of glycoprotein E of TBEV in a region between residues 301-339, which is conserved among flaviviruses from the TBEV group.