Cardiolipin is a hallmark phospholipid of mitochondrial membranes. Despite established significance of cardiolipin in supporting respiratory supercomplex organization, a mechanistic understanding of this lipid-protein interaction is still lacking. To address the essential role of cardiolipin in supercomplex organization, we report cryo-EM structures of a wild type supercomplex (IV1III2IV1) and a supercomplex (III2IV1) isolated from a cardiolipin-lackingSaccharomyces cerevisiaemutant at 3.2-Å and 3.3-Å resolution, respectively, and demonstrate that phosphatidylglycerol in III2IV1occupies similar positions as cardiolipin in IV1III2IV1. Lipid-protein interactions within these complexes differ, which conceivably underlies the reduced level of IV1III2IV1and high levels of III2IV1and free III2and IV in mutant mitochondria. Here we show that anionic phospholipids interact with positive amino acids and appear to nucleate a phospholipid domain at the interface between the individual complexes, which dampen charge repulsion and further stabilize interaction, respectively, between individual complexes.