Ion-membrane interactions are essential to the regulation of cell functions. While numerous molecular dynamics (MD) simulations have been carried out to study the effects of ions on neutral lipid bilayers, few have been conducted on anionic lipid bilayers. Moreover, there is a lack of long-time simulations. Here, submicrosecond MD simulations were performed to investigate the effects of pure cations (K(+), Na(+), and Ca(2+), respectively) on the anionic palmitoyloleoylphosphatidylglycerol (POPG) bilayer first. The results reveal how K(+), Na(+), and Ca(2+) ions influence the structure of anionic lipid bilayers. In general, cations tighten the anionic lipid bilayer and increase the ordering of the lipids. Subsequently, two MD simulations were carried out to elucidate the effects of extra cations added to the bilayers in addition to counterions. It is found that the extra Ca(2+) ions result in stronger effects on the structures of the lipid bilayer, whereas extra Na(+) ions do not. Finally, simulations of ion mixture effects on the structure of the POPG bilayer were conducted, and it is observed that Ca(2+), over K(+) and Na(+), plays a dominant role in affecting the bilayer structures. These results may cast new insights on the distinct functions of Ca(2+) in the biological systems. In addition, our simulations indicate that long-time simulations are necessary to address the effects of ions on lipid bilayer structures.