The mitochondria play a vital role in one of the major apoptosis signalling pathways. Dysfunction in the mitochondria is crucial in the pathogenesis of several viral diseases. In the case of Bovine Viral Diarrhea Virus (BVDV), the cytopathic (CP) biotype, but not the non-cytopathic (NCP) biotype is implicated in the induction of apoptosis. Using state-of-the-art proteomics we demonstrated that bovine monocytes infected with CP BVDV exhibited strong down-regulation of proteins involved in mitochondrial functions, including oxidative phosphorylation- (OxPhos) and the antioxidant catalase (CAT) proteins. In this study, we further assessed the mechanisms of BVDV-induced apoptosis in a susceptible bovine turbinate (BT) cell line by using multiple functional flow cytometry approaches. We demonstrate the direct involvement of CP BVDV but not the NCP counterpart in mitochondrial membrane potential (∆Ψm) disruption and generation of reactive oxygen species (ROS) in infected BT cells. Our data show that CP BVDV contributed to oxidative stress by significantly decreasing the expression of cellular antioxidant enzyme peroxiredoxin1 (PRDX1) and numerically decreasing the levels of CAT. We confirm and extend our proteome findings that the direct damage to mitochondrial proteins decreases their function and contributes to ∆Ψm breakdown and the hyperproduction of ROS seen in BT cells infected with CP but not with NCP BVDV. Overall, our data show that CP and NCP BVDV differentially target mitochondrial proteins and antioxidant enzymes that control the fate of infected cells and determine whether BVDV produces cytopathic effects or replicate non-cytopathically to establish persistent infection.