Interactions between the surface of the intestinal mucus and the molecular components of a food, toxin or drug control the latter's bioabsorption. Understanding the colloidal basis of such interactions requires a thorough thermodynamic characterization of the mucus layer. Towards this aim, surface thermodynamics of porcine intestinal mucin are studied in this work by the aid of inverse gas chromatography (IGC), at infinite dilution and a temperature range (33–60 °C) well below the glass transition. The affinity of several molecular probes, both apolar and polar of different functionalities, onto the mucin surface is evaluated in terms of dispersive and specific interactions calculated by processing the chromatographic retention profiles. Well defined Gaussian peaks, typical of Henry's adsorption, have been obtained for apolar probes. The absence of any thermal transitions is confirmed by the linear drop of the surface free energy with temperature rise, within a typical energy range for carbohydrate polymers (γds = 33–40 mN m−1). For specific polar probes, however, non-Gaussian tailing peaks have been recorded, indicative of desorption retardation phenomena. The potential of polar probes to deploy specific interactions with mucin surface is interpreted on the basis of the homomorph concept. It is inferred that a kind of molecular sieving mechanism assisted by the high polarity of probes seems to favor retention. Evaluation of the surface acid–base interaction potential demonstrates that the mucosal barrier is Lewis amphoteric with predominant basic character. Complementary data obtained via TMDSC, TGA, XRD, and FTIR are discussed in conjunction.