Abstract The pathway for de novo fatty acid biosynthesis begins with the ATP-dependent carboxylation of acetyl-CoA to produce malonyl-CoA. This reaction is catalyzed by acetyl-CoA carboxylase (ACCase), a heteromeric multienzyme complex in bacteria, and in most algae and plants. An emerging theme is the discovery of novel effector proteins that regulate this complex. These effectors include the biotin attachment domain-containing (BADC) and carboxyltransferase interactor (CTI) families, restricted to green algae and plants, as well as PII; each of which is a non-catalytic, negative regulatory effector. These small proteins have a major role in homeostatic control of plastid, heteromeric ACCase (hetACCase) activity and thus fatty acid production. In this review, we summarize the identification and current understanding of the mode of action of these regulatory proteins of the hetACCase. We also discuss knowledge gaps that need to be addressed to obtain a better understanding of the regulation of fatty acid production in autotrophs. Insights into alternative transcript models, and protein phosphorylation of the catalytic subunits and regulatory effectors offer new research directions that may provide novel, targeted engineering approaches that could translate into higher oil crop plants.