Abstract The present work was performed to identify the possible roles of acetyl-CoA carboxylase isoforms (ACC-α and ACC-β). Two forms show 70% amino acid identity, but N-terminal regions share no homology, indicating that these may be uniquely related to the specific role of each ACC form. Thus, we investigated whether introduction of the exogenous ACC N-terminus into H9c2 cardiomyocytes that express both ACC forms causes a noticeable change in a specific pathway of fatty acid metabolism. The effect of ACC-α N-terminus overexpression was specific to the fatty acid synthesis rate resulting in an 80% induction, whereas overexpression of the ACC-β N-terminus increased fatty acid oxidation rate 50% without affecting the fatty acid synthesis rate. These results suggest that ACC-α and β are involved in the regulation of fatty acid synthesis and oxidation, respectively, and that the N-terminus plays an important role in the process. We further demonstrated that novel proteins specifically bound to the ACC N-terminus. This interaction may mediate the involvement of each ACC form in different cellular activities.