Oxidative phosphorylation (OXPHOS) occurs through and across the inner mitochondrial membrane (IMM). Mitochondrial membranes contain a distinct lipid composition, aided by lipid biosynthetic machinery localized in the IMM and class-specific lipid transporters that limit lipid traffic in and out of mitochondria. This unique lipid composition appears to be essential for functions of mitochondria, particularly OXPHOS, by its effects on direct lipid-to-protein interactions, membrane properties, and cristae ultrastructure. This review highlights the biological significance of mitochondrial lipids, with a particular spotlight on the role of lipids in mitochondrial bioenergetics. We describe pathways for the biosynthesis of mitochondrial lipids and provide evidence for their roles in physiology, their implications in human disease, and the mechanisms by which they regulate mitochondrial bioenergetics.