The human telomerase reverse transcriptase (hTERT) is an attractive target for human cancer vaccination because its expression is reactivated in most human tumors. We have evaluated the ability of DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to induce immune responses against hTERT in nonhuman primates (NHPs) (Macaca mulatta). Vaccination was effective in all treated animals, and the adaptive immune response remained detectable and long lasting without side effects. To further enhance the efficacy of the hTERT vaccine, we evaluated the combination of hTERT vaccine and a novel TLR9 agonist, referred to as immunomodulatory oligonucleotide (IMO). Monkeys were dosed weekly with IMO concurrently with the vaccine regimen and showed increases in cytokine secretion and activation of natural killer (NK) cells compared with the group that received vaccine alone. Using a peptide array, a specific profile of B-cell reactive epitopes was identified when hTERT vaccine was combined with IMO. The combination of IMO with hTERT genetic vaccine did not impact vaccine-induced TERT-specific cell-mediated immunity. Our results show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against hTERT with IMO induces multiple effects on innate and adaptive immune responses in NHPs.