Canine cancers occur with an incidence similar to that of humans and share many features with human malignancies including histological appearance, tumor genetics, biological behavior, and response to conventional therapies. As observed in humans, the telomerase reverse transcriptase (TERT) activity is largely confined to tumor tissues and absent in the majority of normal dog tissues. Therefore, dog TERT (dTERT) can constitute a valid target for translational cancer immunotherapy. We have evaluated the ability of adenovirus serotype 6 (Ad6) and DNA electroporation (DNA-EP) to induce immune responses against dTERT in dogs affected by malignant lymphoma (ML). The vaccine was combined with standard chemotherapy regimen [cyclophosphamide, vincristine, prednisone (COP)]. dTERT-specific immune response was induced in 13 out of 14 treated animals (93%) and remained detectable and long-lasting with the absence of autoimmunity or other side effects. Most interestingly, the survival time of vaccine/Chemo-treated dogs was significantly increased over historic controls of Chemo-treated animals (>97.8 versus 37 weeks, respectively, P = 0.001). Our results show that Ad6/DNA-EP-based cancer vaccine against dTERT overcomes host immune tolerance, should be combined with chemotherapy, induces long-lasting immune responses, and significantly prolongs the survival of ML canine patients. These data support further evaluation of this approach in human clinical trials.