Phospholipid scramblase 3 (PLS3) is a newly recognized member of a family of proteins responsible for phospholipid translocation between two lipid compartments. To study PLS3 function in mitochondria, we disrupted its conserved calcium-binding motif yielding an inactive mutant PLS3(F258V). Cells transfected with PLS3(F258V) exhibited reduced proliferative capacity. Mitochondrial analysis revealed that PLS3(F258V)-expressing cells have decreased mitochondrial mass shown by lower cytochrome c and cardiolipin (CL) content, poor mitochondrial respiration, and reduced oxygen consumption and intracellular ATP; whereas wild-type PLS3-transfected cells exhibit increased mitochondrial mass and enhanced respiration. Electron microscopic examination revealed that the mitochondria in PLS3(F258V)-expressing cells have densely packed cristae and are fewer in number and larger than those in control cells. The abnormal mitochondrial metabolism and structure in PLS3(F258V)-expressing cells were associated with decreased sensitivity to UV- and tBid-induced apoptosis and diminished translocation of CL to the mitochondrial outer membrane. In contrast, wild-type PLS3-transfected cells displayed increased sensitivity to apoptosis and enhanced CL translocation. These studies identify PLS3 as a critical regulator of mitochondrial structure and respiration, and CL transport in apoptosis.