Albeit many studies demonstrated that the accumulation of phospholipids in the intestinal mucosal surfaces is essential for the protection of colon epithelia against pathogenic bacteria, the mechanism of interactions between phospholipids and the surface protein mucin is not well understood. In this study, the significance of interfacial interactions between phospholipids and mucin proteins was quantified by the combination of an in vitro intestinal surface model and label-free microinterferometry. The model of intestinal surfaces consists of planar lipid membranes deposited on solid substrates (supported membranes) that display mucin proteins at defined surface densities. Following the quantitative characterization of the systems, we monitored the vertical fluctuation of 10 mu m-large particles on model intestinal surfaces by using microinterferometry, and calculated the effective interfacial interaction potentials by analytically solving the Langevin equation. We found that the spring constant of interfacial potentials calculated based on a harmonic approximation increased concomitantly with the increase in surface potentials, indicating the dominant role of electrostatic interactions. Intriguingly, the spring constants of particles coated with phospholipids do not follow electrostatic interactions. The spring constant of particles coated with zwitterionic phosphatidylcholine was larger compared to membranes incorporating positively or negatively charged lipids. Our data suggested the presence of another underlying molecular level interaction, such as phosphocholine-saccharide interactions. The fact that phosphatidylcholine sustains the binding capability to enzymatically degraded mucin suggests that the direct delivery of phosphatidylcholine to the damaged mucus is a promising strategy for the better treatment of patients affected by inflammatory bowel diseases.