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Spring viraemia of carp virus (SVCV) is a deadly pathogen of common carp. SVCV infection is found to be associated with excess reactive oxygen species (ROS) generation and induces oxidative stress in EPC and FHM cells, which contributes to its pathogenesis. In this study, ROS production and mitochondria function as well as antioxidant enzymes in mitochondria were investigated during SVCV infection in EPC cells. Dysfunction of mitochondria and inactivation of mitochondria electron transport chain complex III to augment O2−ø and H2O2 accumulation were observed in SVCV infected EPC cells. Treatment of Antimycin A reduced the activity of mitochondria complex III in EPC cells, which also inhibited the transcription of SVCV glycoprotein gene (SVCV-G) and production of SVCV. Our studies explain the production of ROS following SVCV infection and also suggest that integrate mitochondrial function is important for SVCV infection.SVCV infection induced mitochondria dysfunction.SVCV infection impairs the activity of mitochondria Complex III to accumulate H2O2 in EPC cells.Integral mitochondrial function is necessary for SVCV infection.