The complex stability constants (KS) and thermodynamic parameters (ΔH° and TΔS°) for the 1:1 complexation of two water‐soluble calixarenes, p‐sulfonatocalix[4]arene (SC4A) and 5,11,17,23‐tetrasulfonato‐25,26,27,28‐tetrakis(n‐butyl)calix[4]arene (SC4A‐Bu), with organic ammonium cations and neutral spherical organic molecules, have been determined by means of isothermal titration calorimetry (ITC) in aqueous solutions at 298.15 K. The obtained results indicate that, upon complexation with these guests by SC4A‐Bu, the enthalpy changes become less favorable, whereas the entropy changes become more favorable relative to SC4A complexation. These differences can be attributed to differential degrees of desolvation and removal of high‐energy water as well as the change in conformation or conformational degrees of freedom upon complexation. The calorimetric investigations, accompanied by 1H NMR and UV/Vis spectroscopy and X‐ray crystallography provide a thermodynamic explanation for the different complexation behavior of SC4A and SC4A‐Bu towards charged and neutral organic guests. Binding ability and molecular selectivity are discussed from the viewpoint of the conformational geometry and electronic properties of hosts and guests.