Despite advances in cancer therapy, cancer related morbidity and mortality among humans and companion animals remains high, and there is a clear need to develop novel targeted therapies. Expression of the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of canine cancer cells and has thus become a candidate for targeted molecular therapies. In this study, the value of telomerase inhibition to target telomerase expressing cancer cells was explored using the novel mechanism of RNA interference (RNAi). Using a Lentiviral expression construct, targeting the RNA component of canine telomerase was effective at inhibiting telomerase in vitro and tumour growth in vivo, but possible resistance mechanisms are highlighted. As canine telomerase biology is more closely related to human telomerase biology than the murine system, it is proposed that this study highlights the value of natural canine models to study anti-telomerase therapies for human patients.