Mitochondrial DNA (
mtDNA) instability activates cGAS-dependent innate immune signaling by unknown mechanisms. Here, we find that
Fanconi anemia suppressor genes are acting in the mitochondria to protect
mtDNA replication forks from instability. Specifically,
Fanconi anemia patient cells show a loss of nascent
mtDNA through MRE11 nuclease degradation. In contrast to DNA replication fork stability, which requires pathway activation by FANCD2-FANCI monoubiquitination and upstream FANC core complex genes, mitochondrial replication fork protection does not, revealing a mechanistic and genetic separation between mitochondrial and nuclear
genome stability pathways. The degraded
mtDNA causes hyperactivation of cGAS-dependent immune signaling resembling the unphosphorylated ISG3 response. Chemical inhibition of MRE11 suppresses this innate immune signaling, identifying MRE11 as a nuclease responsible for activating the
mtDNA-dependent cGAS/
STING response. Collective results establish a previously unknown molecular pathway for
mtDNA replication stability and reveal a molecular handle to control
mtDNA-dependent cGAS activation by inhibiting MRE11 nuclease.