Updated project metadata. The controlled release of mitochondrial content has emerged as a key step in mitochondrial signaling. Particularly the release of mitochondrial DNA (mtDNA) into the cytosol has been shown to activate interferon beta (IFN-β) gene expression to execute the innate immune response. In this report, we show that human adenovirus type 5 (HAdV-C5) induces the release of mtDNA in infected cells. The release of mtDNA is mediated by the viral internal minor capsid protein pVI, which localizes into mitochondria and undergoes mitochondria-specific proteolytic processing. The membrane lytic activity of the pVI and the presence of the mitochondrial membrane proteins Bak and Bax are needed for the mtDNA release. Surprisingly, the pVI-mediate mtDNA release did not increase but blocked IFN-β gene expression. This inhibition was due to the concurrent leakage of the mitochondrial chaperon protein HSP60, which by inhibiting phosphorylation of the interferon regulatory factor 3 (IRF3), blocked IFN-β gene expression. Collectively, our study suggests that the complex release of mtDNA and mitochondrial proteins modulate the IFN-β signaling cascade during pathogenic HAdV-C5 infection.