Acetyl-CoA carboxylase (ACC) catalyses the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Bacterial and most plant chloroplasts contain a multi-subunit ACC (MS-ACC) enzyme that is readily dissociated into its component proteins. Mammals, fungi, and plant cytosols contain the second type of ACC, a single large multifunctional polypeptide. This review will focus on the structures, regulation, and enzymatic mechanisms of the bacterial and plant MS-ACCs.