Cardiolipin (CL), the signature phospholipid of mitochondria, carries four fatty acids that are remodeled after de novo synthesis. In yeast, remodeling is accomplished by the joint action of Cld1, a lipase that removes a fatty acid from CL, and Taz1, a transacylase that transfers a fatty acid from another phospholipid to monolysocardiolipin (MLCL). While taz1 homologs have been identified in all eukaryotes, cld1 homologs have remained obscure. Here, we demonstrate that α/β-hydrolase domain 18 (ABHD18), a highly conserved protein of plants, animals, and humans, is functionally homologous to Cld1. Knockdown of Abhd18 decreased the concentration of MLCL in murine, Taz-knockout myoblasts. Inactivation of Abhd18 in Drosophila substantially increased the abundance of CL. Abhd18 inactivation also reversed the increase in the rate of CL degradation, as measured with C isotopes, and the accumulation of deacylated CLs, such as MLCL and dilyso-CL, in tafazzin (TAZ)-deficient flies. CL species with more than five double bonds were resistant to ABHD18. Our data demonstrate that ABHD18 is the elusive lipase that hydrolyzes CL in mice and flies and presumably in other organisms. Rather than removing just one fatty acid, we show that ABHD18 deacylates CL further. Thus, ABHD18 catalyzes the breakdown of CL, whereas TAZ protects CL from degradation.