JC virus encodes an important regulatory protein, known as Agnoprotein (Agno). We have recently reported Agno's first protein-interactome with its cellular partners revealing that it targets various cellular networks and organelles, including mitochondria. Here, we report further characterization of the functional consequences of its mitochondrial targeting and demonstrated its co-localization with the mitochondrial networks and with the mitochondrial outer membrane. The mitochondrial targeting sequence (MTS) of Agno and its dimerization domain together play major roles in this targeting. Data also showed alterations in various mitochondrial functions in Agno-positive cells; including a significant reduction in mitochondrial membrane potential, respiration rates and ATP production. In contrast, a substantial increase in ROS production and Ca2+ uptake by the mitochondria were also observed. Finally, findings also revealed a significant decrease in viral replication when Agno MTS was deleted, highlighting a role for MTS in the function of Agno during the viral life cycle.