The metabolite acetyl-CoA is necessary in almost all organic life. Cytosolic acetyl-CoA is crucial for lipid synthesis, cholesterol synthesis, and gene regulation. It is also a major factor in metabolic disorders, including diabetes, heart disease, and cancer. In metazoans, the predominant source of cytosolic acetyl-CoA is the essential enzyme ATP-citrate lyase (ACLY). This enzyme is a prime pharmacological target. However, the molecular mechanisms of ACLY activity and regulation are unclear. This work uses a suite of biochemical and biophysical approaches to elucidate ACLY quaternary structure and the mechanisms of acetyl-CoA synthesis. We demonstrate that ACLY forms a tetramer through the previously-uncharacterized ACLY C-terminus. The C-terminus, in the context of the tetramer, interacts with the rest of the protein to bind the reaction substrates. This is a new model for ACLY substrate binding and enzyme activity. We also explore the direct interaction of ACLY with protein acetyltransferases as a means of gene regulation. These findings have implications for our basic understanding of protein biology, evolution, and the rational design of life-saving ACLY therapies.