BackgroundNucleus pulposus cell (NPC) death and progressive reduction play important roles in intervertebral disc degeneration (IVDD). As part of a damage-associated molecular pattern, mitochondrial DNA (mtDNA) can be recognized by TLR9 and triggers the expression of NF-κB and NLRP3 inflammasomes, inducing pyroptosis and inflammatory response. However, whether mtDNA induces NPC pyroptosis via the TLR9-NF-κB-NLRP3 axis and promotes IVDD remains uncertain.MethodsWe constructed an in vitro NPC oxidative stress injury model to clarify the mechanism of mtDNA release, TLR9-NF-κB signaling pathway activation, and NPC injury. We further verified the mechanism of action underlying the inhibition of mtDNA release or TLR9 activation in NPC injury in vitro. We then constructed a rat punctured IVDD model to understand the mechanism inhibiting mtDNA release and TLR9 activation in IVDD.ResultsWe used human NP specimen assays to show that the expression levels of TLR9, NF-κB, and NLRP3 inflammasomes correlated with the degree of IVDD. We demonstrated that mtDNA mediated TLR9-NF-κB-NLRP3 axis activation in oxidative stress-induced human NPC pyroptosis in vitro. Oxidative stress can damage the mitochondria of NPCs, causing the opening of the mitochondrial permeability transition pores (mPTP) and leading to the release of mtDNA into the cytosol. Furthermore, inhibition of mPTP opening or TLR9 activation blocked TLR9-NF-κB-NLRP3 axis activation and thereby mediated NPC pyroptosis and IVDD.ConclusionmtDNA plays a key role in mediating NPC pyroptosis and IVDD via the TLR9-NF-κB-NLRP3 axis. Our findings provide new potential targets for IVDD.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-023-04266-5.