Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here a binding model for the native peptide agonist PYY to the human Y2 receptor based on computational and in vitro pharmacological analyses. The Molecular Dynamics (MD) simulations yielded a conserved binding orientation for the full PYY and five analogues truncated at the amino terminus, which were pharmacologically characterized. The subsequent mutagenesis studies focused on the area binding the common NPY/PYY C-terminal fragment, 32TRQRY36-amide. Several combinations of receptor mutants and PYY-(3-36) analogues modified at either of residues T32, Q34 and the amide group, were designed and tested in binding assays. The results were interpreted by means of MD and Free Energy Perturbation (FEP) simulations of selected mutants. Our results provide a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y2 receptor, which can be used as a basis for optimization of Y2 receptor agonists.