Human cytomegalovirus infection perturbs multiple cellular processes that could promote the release of proapoptotic stimuli. Consequently, it encodes mechanisms to prevent cell death during infection. Using rotenone, a potent inhibitor of the mitochondrial enzyme complex I (reduced nicotinamide adenine dinucleotide- ubiquinone oxido-reductase), we found that human cytomegalovirus infection protected cells from rotenone-induced apoptosis, a protection mediated by a 2.7-kilobase virally encoded RNA (β2.7). During infection, β2.7 RNA interacted with complex I and prevented the relocalization of the essential subunit genes associated with retinoid/interferon-induced mortality-19, in response to apoptotic stimuli. This interaction, which is important for stabilizing the mitochondrial membrane potential, resulted in continued adenosine triphosphate production, which is critical for the successful completion of the virus' life cycle. Complex I targeting by a viral RNA represents a refined strategy to modulate the metabolic viability of the infected host cell.