Hepatitis C virus (HCV) infection induces a state of oxidative stress that is more pronounced than that in many other inflammatory diseases. In this study we used well-characterized cell lines inducibly expressing the entire HCV open-reading frame to investigate the impact of viral protein expression on cell bioenergetics. It was shown that HCV protein expression has a profound effect on cell oxidative metabolism, with specific inhibition of complex I activity, depression of mitochondrial membrane potential and oxidative phosphorylation coupling efficiency, increased production of reactive oxygen and nitrogen species, as well as loss of the Pasteur effect. Importantly, all these effects were causally related to mitochondrial calcium overload, as inhibition of mitochondrial calcium uptake completely reversed the observed bioenergetic alterations. CONCLUSION: Expression of HCV proteins causes deregulation of mitochondrial calcium homeostasis. This event occurs upstream of further mitochondrial dysfunction, leading to alterations in the bioenergetic balance and nitro-oxidative stress. These observations provide new insights into the pathogenesis of hepatitis C and may offer new opportunities for therapeutic intervention.