Arabidopsis (Arabidopsis thaliana), like most dicotyledonous plants, expresses a multicomponent, heteromeric acetyl-CoA carboxylase (htACCase), which catalyzes the generation of the malonyl-CoA precursor of de novo fatty acid biosynthesis. This enzyme consists of four catalytic subunits: biotin carboxylase (BC), carboxyltransferase (CT)-α, CT-β, and biotin carboxyl carrier protein (BCCP1 or BCCP2). By coexpressing combinations of components in a bacterial expression system, we demonstrate noncatalytic BADCs facilitate the assembly and activation of BCCP-BADC-BC subcomplexes catalyzing the bicarbonate-dependent hydrolysis of ATP, which is the first half-reaction catalyzed by the htACCase enzyme. Although BADC proteins do not directly impact formation of the CT-αβ subcomplex, the BADC-facilitated BCCP-BADC-BC subcomplex can more readily interact with the CT-αβ subcomplex to facilitate the generation of malonyl-CoA. The Arabidopsis genome encodes three BADC isoforms (BADC1, BADC2, and BADC3), and BADC2 and BADC3 (rather than BADC1), in combination with BCCP1, best support this quaternary-structural organization and catalytic activation of the htACCase enzyme. Physiological genetic studies validate these attributes as Arabidopsis double mutants singularly expressing BADC2, BADC3, or BADC1 present increasingly greater deleterious impacts on morphological and biochemical phenotypes. Specifically, plants expressing only BADC2 develop normally, plants only expressing BADC3 suffer a stunted root-growth phenotype, and plants expressing only BADC1 are embryo-lethal. The latter phenotype may also be associated with the distinct suborganelle localization of BADC1 in plastids as compared to the localization of the other two BADC homologs. These finding can inspire novel strategies to improve the biological sources of fats and oils for dietary and industrial applications.