Background <p>Intestinal ischemia reperfusion (IIR) is a challenging and life-threatening clinical condition, with disease progression closely linked to excessive inflammatory responses. As a potent activator of innate immunity, the mechanism underlying mitochondrial DNA (mtDNA) release across the mitochondrial membrane remains incompletely elucidated.</p> Methods <p>In this study, an in vivo IIR model was established by clamping the superior mesenteric artery in male mice, and an in vitro hypoxia reoxygenation (HR) model was constructed using Caco-2 cells. Combining multiple techniques including RNA sequencing, subcellular organelle isolation, laser confocal imaging, siRNA transfection, protein cross-linking, Western blotting, enzyme-linked immunosorbent assay (ELISA), and quantitative real-time PCR (qPCR), the regulatory mechanisms of mtDNA release and its biological effects in IIR were systematically verified.</p> Results <p>We found that IIR significantly induced an increase in cytosolic and circulating mtDNA levels, correlating with inflammatory cytokine production. Mechanistic studies revealed that calcium overload mediated by the mitochondrial calcium uniporter (MCU) triggered the opening of the mitochondrial permeability transition pore (mPTP). Meanwhile, the pro-apoptotic protein BAX was recruited to mitochondria and interacted with BAK to form outer mitochondrial membrane oligomeric pores. Notably, although mPTP opening was independent of the BAX/BAK pathway, the two pathways exhibited sequential synergistic effects during mtDNA release. Inhibition of either pathway significantly reduced mtDNA release, decreased inflammatory cytokine levels, and alleviated intestinal tissue injury caused by IIR.</p> Conclusions <p>These findings identify mtDNA as a potential biomarker for IIR and highlight the MCU-mPTP-BAX/BAK axis as a therapeutic target.</p> Graphical abstract <p></p>