The levels of cellular reactive oxygen species (ROS) and ATP as well as the mitochondrial membrane potential (MMP) in response to bovine herpesvirus 1 (BHV-1) infection of MDBK cells were measured, respectively. BHV-1 infection increased ROS production which depended on viral entry, and de novo protein expression and/or DNA replication. Vice versa, excessive ROS was required for efficient viral replication. Levels of both ATP and MMP were significantly decreased after BHV-1 infection. Interestingly, the loss of MMP was ameliorated by ROS depression. Collectively, ROS dependent mitochondrial damage and ultimately disruption of energy metabolism (ATP depletion) are a potential pathogenic mechanism for BHV-1 infection.