Background and Aims Sam50, a key component of the sorting and assembly machinery (SAM) complex, is also involved in bridging mitochondrial outer‐membrane and inner‐membrane contacts. However, the physiological and pathological functions of Sam50 remain largely unknown. Approach and Results Here we show that Sam50 interacts with MICOS (mitochondrial contact site and cristae organizing system) and ATAD3 (ATPase family AAA domain‐containing protein 3) to form the Sam50‐MICOS‐ATAD3‐mtDNA axis, which maintains mtDNA stability. Loss of Sam50 causes mitochondrial DNA (mtDNA) aggregation. Furthermore, Sam50 cooperates with Mic60 to bind to cardiolipin, maintaining the integrity of mitochondrial membranes. Sam50 depletion leads to cardiolipin externalization, which causes mitochondrial outer‐membrane and inner‐membrane (including crista membrane) remodeling, triggering Bax mitochondrial recruitment, mtDNA aggregation, and release. Physiologically, acetaminophen (an effective antipyretic and analgesic)–caused Sam50 reduction or Sam50 liver‐specific knockout induces mtDNA release, leading to activation of the cGAS‐STING pathway and liver inflammation in mice. Moreover, exogenous expression of Sam50 remarkably attenuates APAP‐induced liver hepatoxicity. Conclusions Our findings uncover the critical role of Sam50 in maintaining mitochondrial membrane integrity and mtDNA stability in hepatocytes and reveal that Sam50 depletion–induced cardiolipin externalization is a signal of mtDNA release and controls mtDNA‐dependent innate immunity.

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