Ion mixtures are prevalent in both cytosol and the exterior of a plasma membrane with variable compositions and concentrations. Although abundant MD simulations have been performed to study the effects of single ion species on the structures of lipid bilayers, our understanding of the influence of the ion mixture on membranes is still limited; for example, the competition mechanism of different ions in binding with lipids is not clearly addressed yet. Here, microsecond MD simulations were carried out to study the effects of the mixtures of Ca2+, Mg2+, Na+, and K+ ions on a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer. It has been revealed that the binding efficiency of these ions with POPG lipids is in the following order, Ca2+ > Mg2+ > Na+ > K+. The binding free energy of Ca2+ to the lipid bilayer is similar to-4.0 kcal/mol, which is much lower than those of other ions. This result explains why the effects of the ion mixture on membranes are particularly sensitive to the concentration of calcium. The on-rates of different ions do not have a large difference, while the off-rate of Ca2+ is 2-3 orders of magnitude smaller than those of the others. Therefore, the strongest binding affinity of Ca2+ is mainly determined by its smallest off-rate. In addition, our study suggests that the structure of the lipid bilayer is influenced dominantly by the concentration of Ca2+ ions. The simulation results also provide a good explanation for a variety of biological processes relevant to Ca2+ and Mg2+ regulations, such as membrane fusion.