In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K + , Mg 2+ , and Ca 2+ ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca 2+ ions with lipids is significantly stronger than that of K + and Mg 2+ ions, regardless of the composition of the lipid bilayer. The binding of Ca 2+ ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K + - and Mg 2+ -containing systems. The Ca 2+ ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength—most notably for Ca 2+ . The formation of cation–lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca 2+ –phospholipid clusters across apposed lipid bilayers can work as a “cation glue” to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.