Iron cations are indispensable players in a number of vital biological processes such as respiration, cell division, nitrogen fixation, oxygen transport, nucleotide synthesis, oxidant protection, O-2 activation in the metabolism of various organic substrates, gene regulation, and protein structure stabilization. The basic mechanisms and factors governing the competition between Fe2+ and other metal species from the cellular fluids such as Mg2+, Mn2+, and Zn2+ are, however, not well understood, and several outstanding questions remain. (i) How does the Fe2+ binding site select the right cation and protect itself from attacks by other biogenic cations present in the surrounding milieu? (ii) Do the iron binding sites employ different selectivity strategies toward metal cations possessing different ligand affinities and cytosolic concentrations? (iii) What are the key determinants of metal selectivity in Fe2+ proteins? In this study, by employing density functional theory calculations combined with polarizable continuum model computations, we endeavor to address these questions by evaluating the thermodynamic outcome of the competition between Fe2+ and Mg2+/Mn2+/Zn2+ in model non-heme mononuclear metal binding sites of various compositions and charge states. The present calculations, which are in line with available experimental data, shed light on the mechanism of Fe2+Mg2+/Mn2+/Zn2+ competition in non-heme iron proteins and disclose the key factors governing the process.