Infection with the challenge virus standard-11 (CVS) strain of fixed rabies virus induces neuronal process degeneration in adult mice after hindlimb footpad inoculation. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal protein adduct staining, indicating a critical role of oxidative stress. Mitochondrial dysfunction is the major cause of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection significantly increased maximal uncoupled respiration and complex IV respiration and complex I and complex IV activities, but did not affect complex II-III or citrate synthase activities. Increases in complex I activity, but not complex IV activity, correlated with susceptibility of the cells to CVS infection. CVS infection maintained coupled respiration and rate of proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by a high NADH/NAD+ ratio. The basal production of reactive oxygen species (ROS) was not affected in CVS-infected neurons. However, a higher rate of ROS generation occurred in CVS-infected neurons in the presence of mitochondrial substrates and inhibitors. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration and oxidative stress.