A better understanding of the neutralizing epitopes may facilitate the generation of new antibody-based therapeutics against DENV infection. In this study, we generated several mAbs against DENV-2. We found that serotype-specific anti-E-DIII mAbs played an important role in the neutralization of virus infectivity. Studies of the neutralizing epitopes found the strongest mAbs to be DB32-6 and DB25-2, both DENV-2 serotype-specific antibodies. These two mAbs recognized the A-strand of E-DIII at residues K310 and E311, respectively. Humanized DB32-6 mAb efficiently neutralized DENV-2 infection in a therapeutic mouse model and its variant version prevented enhancing activity.