Cardiolipin (CL) is a unique phospholipid that is almost exclusively localized at the level of the inner mitochondrial membrane (IMM), where it is biosynthesized. This phospholipid is associated with membranes which are designed to generate an electrochemical gradient that is used to produce ATP. Such membranes include the bacterial plasma membrane and IMM. This ubiquitous and intimate association between CL and energy-transducing membranes suggests an important role for CL in mitochondrial bioenergetic processes. CL has been shown to interact with a number of IMM proteins, including the respiratory chain complexes and substrate carriers. Moreover, CL is involved in different stages of the mitochondrial apoptosis process as well as in mitochondrial membrane stability and dynamics. Alterations in CL structure, content, and acyl chain composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions and aging. In this review, we provide an overview of the roles of CL in mitochondrial function and bioenergetics in health and disease. Antioxid. Redox Signal. 20, 1925–1953.