Dengue virus (DENV) infection is an expanding global threat to public health. Effective vaccine and treatment approaches remain elusive. B cell-directed vaccines may be complicated by an antibody-dependent enhancement (ADE) phenomenon based on cross-serotype, nonneutralizing antibodies. We hypothesized that a CD8(+) T cell-directed genetic vaccine that targets a DENV nonstructural protein, NS1, could be a potential strategy to overcome the ADE barrier and accomplish cross-serotype protection. We selected an adenovirus-based vector as the dengue vaccine carrier. To bypass preexisting immunity to human adenoviruses and to improve vaccine efficacy, we created vaccine vectors, based on simian adenoviruses SAdV22 and SAdV25 as well as human adenovirus serotype 5, expressing the NS1 antigen of the Hainan strain, DENV serotype 2. An NS1 peptide library was screened to identify the immunodominant and functional epitope within the NS1 protein for H-2(d)-restricted CD8(+) T cells in BALB/c mice, using interferon-gamma enzyme-linked immunospot and intracellular cytokine-staining assays. Our study identified the 9-mer AGPWHLGKL (NS1(265273)) as the H-2(d)-restricted T cell epitope whose sequence is highly conserved among 26 strains of DENV serotypes 1, 2, 3, and 4, suggesting potential cross-serotype protection of NS1-directed genetic vaccines in the BALB/c model of DENV infection. Importantly, we characterized the cytokine profile of CD8(+) NS1-specific T cells in BALB/c mice vaccinated with HAdV-5-NS1, SAdV-22-NS1, and SAdV-25 NS1 and demonstrated the effective in vivo cytolytic killing capacity of CD8(+) T cells from SAdV-25-NS1-vaccinated mice.