The article reviews the role of mitochondrial DNA (mtDNA) in activating innate immune signaling pathways and the crosstalk among these pathways. MtDNA, a damage-associated molecular pattern (DAMP), activates Toll-like receptor 9 (TLR9), NLRP3 inflammasome, and cGAS-STING signaling pathways. These pathways are interconnected and play crucial roles in antimicrobial defense, antiviral signaling, and inflammatory diseases. The release of mtDNA into the cytoplasm can occur through various mechanisms, including mitochondrial outer membrane permeabilization (MOMP) and the mitochondrial permeability transition pore (mTPP). MtDNA activates TLR9, leading to the production of inflammatory factors and interferons. It also activates the NLRP3 inflammasome, causing pyroptosis and inflammation. Additionally, mtDNA activates the cGAS-STING pathway, which induces type I interferon responses and pro-inflammatory cytokines. The crosstalk between these pathways is complex and involves multiple signaling molecules and pathways. The article highlights the importance of understanding these interactions for the development of strategies to prevent and treat inflammatory diseases mediated by mtDNA.The article reviews the role of mitochondrial DNA (mtDNA) in activating innate immune signaling pathways and the crosstalk among these pathways. MtDNA, a damage-associated molecular pattern (DAMP), activates Toll-like receptor 9 (TLR9), NLRP3 inflammasome, and cGAS-STING signaling pathways. These pathways are interconnected and play crucial roles in antimicrobial defense, antiviral signaling, and inflammatory diseases. The release of mtDNA into the cytoplasm can occur through various mechanisms, including mitochondrial outer membrane permeabilization (MOMP) and the mitochondrial permeability transition pore (mTPP). MtDNA activates TLR9, leading to the production of inflammatory factors and interferons. It also activates the NLRP3 inflammasome, causing pyroptosis and inflammation. Additionally, mtDNA activates the cGAS-STING pathway, which induces type I interferon responses and pro-inflammatory cytokines. The crosstalk between these pathways is complex and involves multiple signaling molecules and pathways. The article highlights the importance of understanding these interactions for the development of strategies to prevent and treat inflammatory diseases mediated by mtDNA.