We studied the thermodynamics of binding of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X_PG, by means of isothermal titration calorimetry. So-called lipid-to-peptide and peptide-to-lipid titration experiments were performed. The experimental data were interpreted in terms of the surface partitioning model. Membrane binding is driven by an exothermic partial molar enthalpy of −(20–30) kJ mol⁻¹ owing to the nonclassical hydrophobic effect and a lipid-induced change of the secondary structure of penetratin from a random coil into a more ordered α-helical and/or β-sheet conformation. The differential binding enthalpy slightly changes as a function of the content of anionic lipid in the membrane and of the molar ratio bound peptide-to-lipid. This effect presumably reflects variations of the secondary structure of bound penetratin. The small change of entropy upon binding is compatible with a superficial binding mode of the peptide with relatively small perturbations of the membrane.