Oxidized mitochondrial DNA (mtDNA) activates the NLRP3 inflammasome during apoptosis. The study shows that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome is activated by mitochondrial apoptotic signaling, leading to the production of interleukin-1β (IL-1β). Oxidized mtDNA released into the cytosol during apoptosis binds to and activates the NLRP3 inflammasome. The anti-apoptotic protein Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induces NLRP3 inflammasome activation, as macrophages lacking mtDNA have severely attenuated IL-1β production. Oxidized mtDNA binding to the NLRP3 inflammasome and IL-1β secretion can be competitively inhibited by the oxidized nucleoside 8-OH-dG. These findings reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome, providing a missing link between apoptosis and inflammasome activation via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. The study also shows that mitochondrial dysfunction and apoptosis are closely linked to ROS, intracellular K+, and lysosomal degradation, positioning mitochondria as the central hub for integrating diverse signals sensed by the NLRP3 inflammasome. The results indicate that apoptotic signals activate the NLRP3 inflammasome through the release of oxidized mtDNA, which binds to and activates the inflammasome. Bcl-2 expression regulates NLRP3 inflammasome activation and IL-1β secretion. Apoptosis is necessary for NLRP3 inflammasome activation, as Bcl-2 overexpression reduces IL-1β secretion. The study also demonstrates that mtDNA is required for NLRP3 inflammasome-mediated IL-1β secretion. Oxidized mtDNA binds to NLRP3 and activates the inflammasome, while oxidized nucleoside 8-OH-dG competitively inhibits this interaction. The findings highlight the role of oxidized mtDNA in NLRP3 inflammasome activation during apoptosis.Oxidized mitochondrial DNA (mtDNA) activates the NLRP3 inflammasome during apoptosis. The study shows that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome is activated by mitochondrial apoptotic signaling, leading to the production of interleukin-1β (IL-1β). Oxidized mtDNA released into the cytosol during apoptosis binds to and activates the NLRP3 inflammasome. The anti-apoptotic protein Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induces NLRP3 inflammasome activation, as macrophages lacking mtDNA have severely attenuated IL-1β production. Oxidized mtDNA binding to the NLRP3 inflammasome and IL-1β secretion can be competitively inhibited by the oxidized nucleoside 8-OH-dG. These findings reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome, providing a missing link between apoptosis and inflammasome activation via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. The study also shows that mitochondrial dysfunction and apoptosis are closely linked to ROS, intracellular K+, and lysosomal degradation, positioning mitochondria as the central hub for integrating diverse signals sensed by the NLRP3 inflammasome. The results indicate that apoptotic signals activate the NLRP3 inflammasome through the release of oxidized mtDNA, which binds to and activates the inflammasome. Bcl-2 expression regulates NLRP3 inflammasome activation and IL-1β secretion. Apoptosis is necessary for NLRP3 inflammasome activation, as Bcl-2 overexpression reduces IL-1β secretion. The study also demonstrates that mtDNA is required for NLRP3 inflammasome-mediated IL-1β secretion. Oxidized mtDNA binds to NLRP3 and activates the inflammasome, while oxidized nucleoside 8-OH-dG competitively inhibits this interaction. The findings highlight the role of oxidized mtDNA in NLRP3 inflammasome activation during apoptosis.