Ions play a key role in mediating interactions between cytoskeletal proteins, helping to govern the assembly process and mechanical properties of actin filaments, which are composed of negatively charged monomers. Recent work using structural bioinformatics and site-specific mutagenesis suggests the existence of two specific divalent cation binding sites, one of which participates in the actin polymerization process, while the other modulates actin filament stiffness and plays a role in cofilin-mediated filament severing. To date, structural methods have not been able to resolve cation-associated filaments, and so in this work we turn to molecular dynamics simulations to predict candidate binding pocket geometries and to elucidate the mechanism by which binding in the stiffness site effects a filament's mechanical properties. We find that little change is necessary to an actin monomer conformation in order to incorporate an additional magnesium ion in the “polymerization site,” positioned in the long axis between actin monomers. We furthermore show that binding of an additional magnesium ion in the “stiffness site” adheres the actin D-loop to the adjacent actin's target binding cleft, resulting in a decrease in torsional flexibility and an increase in filament persistence length, consistent with previous biochemical and biophysical studies. Given these candidate structures, our simulations allow us to dissect the mechanism for this stiffening by studying the conformational space accessible to the D-loop. We also compute the burial of certain key conserved residues in the D-loop with and without coordinated magnesium ions, and hence predict their accessibility to enzymes known to regulate actin filament dynamics by post-translational modification of these amino-acids.