Protein palmitoylation is a widespread lipid modification in which one or more cysteine thiols on a substrate protein are modified to form a thioester with a palmitoyl group. This lipid modification is readily reversible; a feature of protein palmitoylation that allows for rapid regulation of the function of many cellular proteins. Mutations in palmitoyltransferases (PATs), the enzymes that catalyze the formation of this modification, are associated with a number of neurological diseases and cancer progression. This review summarizes the crucial role of palmitoylation in biological systems, the discovery of the DHHC protein family that catalyzes protein palmitoylation, and the development of methods for investigating the catalytic mechanism of PATs.