Significance Protein palmitoylation is one of the most highly abundant protein modifications, through which long-chain fatty acids get attached to cysteines by a thioester linkage. It plays critically important roles in growth signaling, the organization of synaptic receptors, and the regulation of ion channel function. Yet the molecular mechanism of the DHHC family of integral membrane enzymes that catalyze this modification remains poorly understood. Here, we present the structure of a precatalytic complex of human DHHC20 with palmitoyl CoA. Together with the accompanying functional data, the structure shows how a bivalent recognition of palmitoyl CoA by the DHHC enzyme, simultaneously at both the fatty acyl group and the CoA headgroup, is essential for catalytic chemistry to proceed.