PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.