Mitochondrial DNA (mtDNA) stress primes the antiviral innate immune response. This study shows that moderate mtDNA stress, induced by the loss of the mtDNA packaging protein TFAM, activates cytosolic antiviral signaling pathways, leading to the expression of interferon-stimulated genes (ISGs) and enhanced type I interferon responses. Mechanistically, aberrant mtDNA packaging leads to the release of mtDNA into the cytosol, where it is sensed by the DNA sensor cGAS, triggering STING-IRF3-dependent signaling that elevates ISG expression and enhances viral resistance. The study also demonstrates that herpesviruses induce mtDNA stress, which potentiates antiviral signaling and type I interferon responses during infection. These findings highlight the role of mitochondria in innate immunity and identify mtDNA stress as a cell-intrinsic trigger of antiviral signaling. The study further shows that mtDNA homeostasis monitoring cooperates with canonical virus sensing mechanisms to fully license antiviral innate immunity. The results indicate that mitochondrial stress directly potentiates antiviral innate immunity, and that herpesvirus-induced mtDNA stress is necessary for effective engagement of ISG expression and antiviral priming. The study provides a novel understanding of how mtDNA stress activates the antiviral innate immune response, revealing a critical role for mitochondria in innate immunity and suggesting that mitochondrial dysregulation may contribute to the pathogenesis of human diseases and aging.Mitochondrial DNA (mtDNA) stress primes the antiviral innate immune response. This study shows that moderate mtDNA stress, induced by the loss of the mtDNA packaging protein TFAM, activates cytosolic antiviral signaling pathways, leading to the expression of interferon-stimulated genes (ISGs) and enhanced type I interferon responses. Mechanistically, aberrant mtDNA packaging leads to the release of mtDNA into the cytosol, where it is sensed by the DNA sensor cGAS, triggering STING-IRF3-dependent signaling that elevates ISG expression and enhances viral resistance. The study also demonstrates that herpesviruses induce mtDNA stress, which potentiates antiviral signaling and type I interferon responses during infection. These findings highlight the role of mitochondria in innate immunity and identify mtDNA stress as a cell-intrinsic trigger of antiviral signaling. The study further shows that mtDNA homeostasis monitoring cooperates with canonical virus sensing mechanisms to fully license antiviral innate immunity. The results indicate that mitochondrial stress directly potentiates antiviral innate immunity, and that herpesvirus-induced mtDNA stress is necessary for effective engagement of ISG expression and antiviral priming. The study provides a novel understanding of how mtDNA stress activates the antiviral innate immune response, revealing a critical role for mitochondria in innate immunity and suggesting that mitochondrial dysregulation may contribute to the pathogenesis of human diseases and aging.