Abstract Body: Background Barth syndrome (BTHS) is a disorder caused by mutations in the TAFAZZIN gene, which disrupts cardiolipin (CL) metabolism and leads to cardiac and skeletal myopathy, neutropenia, and 3-methylglutaconic aciduria. Altered CL impairs mitochondrial function, contributing to symptoms of BTHS. Despite extensive research, the molecular mechanisms underlying BTHS remain unclear, and no cure exists. Mitochondrial protein quality control (MPQC) is crucial for maintaining mitochondrial homeostasis, yet its role in BTHS is underexplored. Our preliminary data indicate elevated levels of Caseinolytic peptidase B (CLPB), a mitochondrial chaperonin localized in the intermembrane space (IMS) with disaggregase activity, in BTHS patient-derived cells, and Tafazzin-deficient BTHS mice. Hypothesis Our data-driven hypothesis proposes that CLPB regulates the quality control of IMS proteins, mitochondrial function, and bioenergetics to support cardiac health. However, this adaptive response becomes maladaptive during BTHS pathology. Aim We aim to understand CLPB’s function in BTHS and explore its potential as a therapeutic target. Methods We generated CLPB knockouts in both control and Tafazzin^-/- C2C12 cells, as well as a CLPB knockdown mouse model in Tafazzin^fl/fl (control) and Tafazzin^fl/fl x aMHC-Cre. Using cellular, molecular, and physiological approaches, we evaluated CLPB’s impact on mitochondrial homeostasis and its therapeutic potential in BTHS. Results CLPB loss improves NADH and calcium retention capacity without affecting mitochondrial respiration rate & membrane potential, ATP, and mtDNA content in Tafazzin^-/--Clpb^-/- compared to control, Clpb^-/- and Tafazzin^-/- C2C12 cells. To explore the underlying mechanisms behind changes in the metabolic state of Tafazzin^-/--Clpb^-/- cells, we analyzed transcripts of CL regulators (ALCLAT1 and CLS1), mitochondrial biogenesis (PGC1a, Tfam, and NRF1/2), and mtUPR (ATF4/5, HSP70, CHOP, and SOD2) markers. The Tafazzin^-/--Clpb^-/- activate mitochondrial biogenesis and CL synthesis by elevating PGC1a and CLS1 transcripts, while their mtUPR transcript profile is comparable to control cells. Furthermore, echocardiographic analysis shows restoration of ejection fraction in CLPB^KD and Tafazzin^fl/fl x aMHC-Cre-CLPB^KD mice. Conclusion These findings highlight CLPB’s crucial role in IMS proteins quality control and mitochondrial homeostasis, providing a basis for new therapeutic avenues against mitochondrial dysfunction in BTHS.