The study investigates the cellular responses to mitochondrial DNA (mtDNA) stress, a condition observed in various human diseases and aging. MtDNA is normally packaged into nucleoids by the abundant mtDNA-binding protein, transcription factor A mitochondrial (TFAM). Complete mtDNA depletion impairs oxidative phosphorylation (OXPHOS) and triggers stress signaling and metabolic responses. However, the cellular responses to mtDNA instability remain unclear. The authors show that moderate mtDNA stress, induced by TFAM deficiency, engages cytosolic antiviral signaling to enhance the expression of a subset of interferon-stimulated genes (ISGs). Mechanistically, aberrant mtDNA packaging promotes the release of mtDNA into the cytosol, where it engages the DNA sensor cGAS and promotes STING-IRF3-dependent signaling, leading to elevated ISG expression and potentiated type I interferon responses. Additionally, herpesviruses induce mtDNA stress, which potentiates antiviral signaling and type I interferon responses during infection. The study demonstrates that mitochondria are central participants in innate immunity and identifies mtDNA stress as a cell-intrinsic trigger of antiviral signaling. It also suggests that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully license antiviral innate immunity.The study investigates the cellular responses to mitochondrial DNA (mtDNA) stress, a condition observed in various human diseases and aging. MtDNA is normally packaged into nucleoids by the abundant mtDNA-binding protein, transcription factor A mitochondrial (TFAM). Complete mtDNA depletion impairs oxidative phosphorylation (OXPHOS) and triggers stress signaling and metabolic responses. However, the cellular responses to mtDNA instability remain unclear. The authors show that moderate mtDNA stress, induced by TFAM deficiency, engages cytosolic antiviral signaling to enhance the expression of a subset of interferon-stimulated genes (ISGs). Mechanistically, aberrant mtDNA packaging promotes the release of mtDNA into the cytosol, where it engages the DNA sensor cGAS and promotes STING-IRF3-dependent signaling, leading to elevated ISG expression and potentiated type I interferon responses. Additionally, herpesviruses induce mtDNA stress, which potentiates antiviral signaling and type I interferon responses during infection. The study demonstrates that mitochondria are central participants in innate immunity and identifies mtDNA stress as a cell-intrinsic trigger of antiviral signaling. It also suggests that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully license antiviral innate immunity.