Cardiolipin (CL) is a phospholipid found exclusively in mitochondria and is required for normal mitochondrial function. CL biosynthesis requires a crucial remodelling step that incorporates specific acyl chains onto its molecular structure. The enzyme primarily responsible for CL remodelling is Tafazzin (TAZ), a mitochondrial protein encoded by the TAZ gene localized to chromosome Xq28.12. Mutations on the TAZ gene result in a rare yet severe disease known as Barth Syndrome (BTHS). BTHS is characterized by symptoms that include cardiomyopathies, neutropenia and skeletal myopathies. The mitochondrial enzyme Monolysocardiolipin Acyltransferase -1 (MLCL AT-1) also exhibits the ability to remodel CL with specific acyl chains. The aims of our study were to 1) determine if a relationship exists between TAZ and MLCL AT-1 , 2) determine if MLCL AT-1 expression in BTHS lymphoblasts leads to improvements in mitochondrial function, and 3) to use the Taz knockdown (KD) mouse model to get a better understanding of the phenotypes displayed by BTHS patients. Our results showed that in normal healthy lymphoblasts, expression of MLCL AT-1 was inversely dependent on TAZ expression. However, in BTHS lymphoblasts, expression of MLCL AT-1 was significantly lower compared to healthy controls. With the use of a MLCL AT-1-carrying plasmid, we expressed MLCL AT-1 in BTHS cells. This resulted in increased MLCL AT-1 gene, protein and enzyme activity. In addition, expression of MLCL AT-1 in BTHS cells resulted in increases in CL mass, improved mitochondrial function and a reduction in reactive oxygen species (ROS) production. However, no changes were detected in mitochondrial respiratory chain supercomplex (SC) assembly in BTHS cells expressing MLCL AT-1 compared to healthy controls. SC formation was disrupted in the hearts and skeletal muscle, but not the liver, of the Taz KD mice compared to wild-type (WT) animals. These results correlated with an elevated generation of hydrogen peroxide (H2O2) in the heart and skeletal muscle mitochondria of Taz KD mice compared to WT. Liver mitochondria from Taz KD mice, on the other hand, generated significantly less H2O2 compared to WT mice. The results from this study and our other published work demonstrate that MLCL AT-1 expression varies depending on the health of mitochondria and is tissue specific. In addition, our results reveal that TAZ expression is essential for various aspects of mitochondrial function including SC formation and ROS production.