Updated project metadata. Cytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remained enigmatic. We found that mtDNA-dependent immune signaling via the cGAS-STING pathway is under metabolic control and induced by cellular nucleotide deficiency. The mitochondrial protease YME1L preserves nucleotide pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33, limiting mitochondrial nucleotide import and accumulation of mtDNA. Deficiency of YME1L or the mitochondrial transcription factor A (TFAM) drives an mtDNA-dependent inflammatory response, which depends on SLC25A33 and is suppressed upon replenishment of cellular nucleotide pools. Metabolic insults that deplete cytosolic nucleotides trigger mtDNA-dependent immune responses. Our results thus identify mtDNA release and innate immune signaling as a metabolic response, offering new therapeutic opportunities in disease.