Dysregulation of free acid metabolism is a major contributor to the development of insulin resistance and diabetes. Mitochondrial trifunctional enzyme subunit (MTPα) has a critical role in fatty acid β-oxidation. However, the association between MTPα and insulin resistance is not definitively known. Here, we aimed to determine how MTPα affects insulin resistance. We tested how MTPα affected glucose uptake in insulin-resistant 3T3-L1 adipocytes and white adipose tissue (WAT) of db/db diabetic mice. We also measured how acetylation and ubiquitylation modifications regulated MTPα activation and stability, using quantitative real-time polymerase chain reactions, immunoblotting, and immunoprecipitation. We found that MTPα overexpression promoted glucose uptake via Glut4 translocation to the plasma membrane in 3T3-L1 adipocytes. Moreover, MTPα upregulation decreased glycemia in db/db mice. Deacetylation increased MTPα protein stability and its ability to reduce insulin resistance. The activation of SIRT1, a major deacetylase, prevented MTPα degradation by decreasing its acetylation in adipocytes. Our study demonstrates a new role for MTPα in reducing insulin resistance. Acetylation and ubiquitylation modifications of MTPα were crucial to regulating its function in glucose metabolism.