Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). BLV can interact with telomerase and inhibits telomere shortening, contributing in leukemogenesis and tumour induction. The role of telomerase in BLV-induced lymphosarcoma and aging has been extensively studied. To date, the interaction of both BLV and aging on telomerase mis-regulation have, however, not been investigated. In the present study, telomerase activity in BLV positive and negative cows was compared over a wide range of ages (11-85 months). Lymphocyte counts were also measured in both BLV positive and negative groups. Telomerase activity was detected in all BLV infected animals with persistent lymphocytosis (PL), especially in older individuals. This study revealed that the cells undergo the natural telomerase shortening even in the presence of an existing viral infection. We also show that viral infection, especially during the PL phase of the disease, increases telomerase activity. A statistically significant interaction between age and viral infection was observed for telomere shortening during BLV infection. Older animals with BLV infection, especially those with persistent lymphocytosis or visible tumors, exhibited a sharp increase in telomerase activity. This study demonstrates that there is a significant interaction between BLV infection and telomerase up-regulation and lymphocytosis.