We report a novel strategy for reversible immobilization of diffusive membrane-associated proteins in a native orientation using a liquid-gel bilayer phase transition, and its application to the single molecule study of Cy3-labeled Annexin V (A5) monomers on supported lipid bilayers containing phosphatidylserine (PS) in a Ca2+-rich environment. Total internal reflection fluorescence single molecule trajectory analysis revealed that, at low membrane occupancy, A5 monomers diffuse randomly on liquid phase bilayers and occasionally collide with other A5 monomers to form short-lived pseudodimers. During the liquid-to-gel bilayer phase transition, diffusive A5 monomers become immobilized mostly as isolated monomers, with some percentage of dimers and trimers. The EDTA-induced unbinding of immobilized Cy3-A5 spots indicates that Ca2+-bridges between A5 and PS lipids are preserved in the immobilized A5 monomers, confirming their native orientation on gel phase bilayers. Furthermore, the persistence of Ca2+-bridges during the liquid-to-gel phase transition, despite negligible A5 binding affinity to gel phase bilayers, strongly suggests the formation of tightly bound A5(Ca2+)(m)(PS)(n) complexes that diffuse and become immobilized as single units during the bilayer phase transition.