Biological membranes are essential for the integrity of cells, providing a barrier between the internal and external environments. They also accommodate a great diversity of proteins, which participate in a wide range of critical biological processes, such as energy or signal transduction, solute transport, cell-cell recognition, and traficking. The physical properties of membranes are important for their barrier function, which can be regulated by the surrounding environmental factors, such as the presence of various molecules and ions. For example, previous studies have shown that the adsorption of Ca 2+ , one of the most important secondary messengers in cells, can inluence many physical properties of membranes, such as the thicknesses, 2 head group orientations of lipids, 2±5 ordering of acyl chains, 3,5±10 lipid dehydration, 2,4±6,8 and lateral diffusion. Phosphatidylcholine (PC) is the most abundant zwitterionic phospholipid in eukaryotic cell membranes and subcellular organelles. Phosphatidylserine (PS) is an anionic phospholipid present in most inner lealets of cellular organelles, including the plasma membrane, where it accounts for as much as 28% of all lipids. 18±21 Therefore, biological membranes are intrinsically asymmetric with different lipid compositions in the inner and outer lealets, and a mixture of PC and PS is often used as a general model for membrane studies. Moreover, the concentration of calcium ions across membranes is often distributed asymmetrically. Intracellularly, the free calcium ion concentration typically ranges between 10 and 100 nM. However, within organelles such as the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR), the concentration can reach signiicantly higher levels, ranging from 60 to 500 µM, and in some cases, even up to 1 mM. In contrast, the extracellular free calcium ion concentration is approximately 1.8 mM. In addition, PS lipids were found to play additional roles by interacting with signaling proteins, regulating surface charges and protein localization, and inducing protein aggregation. Some proteins attract PS lipids by nonspeciic electrostatic interactions, and the binding can be regulated by Ca . Therefore, the interaction between Ca 2+ and the negatively charged PS is of particular interest.