Dengue virus (DENV) is a mosquito-borne disease caused by four genetically and serologically related viruses termed DENV-1, -2, -3 and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2 and ED3. The ED3 domain contains DENV type-specific and DENV complex-reactive antigenic sites. To date, nearly all antigenic studies on the E protein have focused on DENV-2. In this study, the epitope recognized by a DENV-3 type-specific monoclonal antibody (mAb 14A4-8) was mapped to the DENV-3 ED3 domain using a combination of physical and biological techniques. Epitope mapping revealed that amino acid residues V305, L306, K308, E309, V310, K325, A329, G381 and I387 were critical for the binding of mAb 14A4-8 and amino acid residues T303, K307, K386, W389 and R391 were peripheral residues for this epitope. The location of the mAb 14A4-8 epitope overlaps with the DENV complex-reactive antigenic site in the DENV-3 ED3 domain.