The physiochemical properties of the phospholipid bilayer directly modulate the interaction between adhered cells and extracellular matrixes. The thermotropic transition of phospholipid bilayer has been shown to be highly coupled with the mechanical properties of liposomes (Heimburg, T. Biochim. Biophys. Acta 1998, 147, 1415). However, there is currently a lack of understanding of the thermal-induced effect on the adhesive contact mechanics of liposomes adhering on non-deformable substrates. In this study, high-resolution reflectance interference contrast microscopy, phase contrast microscopy, and contact mechanics modeling are applied to probe the interfacial adhesion contacts of phospholipid vesicles on fused silica substrates during gel to liquid crystalline transition of the bilayer. It is shown that the contact area, contact angle, and adhesion energy of adhering liposome increase during the thermotropic transition. The trend of adhesion energy against temperature is dictated by the complex interplay of the elastic modulus of the wall and deformation of the liposome. Besides, adhesion energy decreases when the size of vesicle increases in isotonic condition. In the presence of osmotic stress, the magnitude of adhesion is solely dictated by the elastic modulus and is independent of vesicle size. When the wall thickness of liposome increases, the shape of the contact zone is modified by the thermal effect despite the constant contact area. Overall, this study provides direct evidence that the adhesion strength of liposome can be tuned by the change of temperature, sheds light on the physical origin of the thermal-induced process, and may explain why anchorage-dependent cells must be maintained at a particular temperature.