Peroxisome proliferator-activated receptors (PPARs) belong to the members of the nuclear receptor superfamily, and play important roles in lipid and glucose homeostasis. Residue Phe282 in PPARgamma (Phe273 in PPARalpha), beyond the ligand-binding site, is a conserved amino acid across several nuclear receptors and in all PPAR subfamily. In this work, we firstly investigated the influence of Phe282(273)Ala mutation on the binding affinity of PPARgamma(alpha) against a series of agonists by use of surface plasmon resonance (SPR) technique and cellular transcriptional activation analysis. Phe282(273)Ala mutation decreases the binding affinities of the ligands to the receptors in certain degrees, from several to 1000-folds. Phe282Ala mutation dramatically reduced the binding affinity of PPARgamma to GI262570, however, this mutation did not affect PPARalpha binding to this ligand, thereby suggesting that the Phe282 and Phe273 are associated with the selectivity of GI262570 binding to PPARgamma and PPARalpha. The mutation reduced the transcriptional activation activities of the receptors induced by the ligand binding, and the decrease degree is generally in agreement with the binding affinities of the ligands to the receptors. The 5 ns MD simulations for the wild-type and mutated PPARgamma showed that the mutation did not influence the flexibility of the receptor. There is no repulsion between Phe282 and the proceeding loop of AF2. However, substitution of Phe282 by alanine enlarged the entrance of the binding pocket and abolished the repulsive interaction between solvent water molecules and this hydrophobic residue, thus more water molecules can enter into the binding pocket. It needs more energy to exclude the extra water molecules for a ligand binding to the mutated receptor. In addition, the extra water molecules abolish some of H-bonds between the ligand and receptors. Therefore, solvent effect may be concluded as the major source of the decrease of binding affinity for the mutated receptors to the ligands, and thereby of the decrease of their transcriptional activation activities.