The sarcoplasmic reticulum (SR) Ca2+-ATPase, a P-type transmembrane protein, can transport Ca2+ from the cytoplasmic to the luminal side over other cations specifically. The proposed Ca2+ entrance channel, composed of the main-chain carbonyl oxygen and side-chain carboxyl oxygen atoms of the amino acids, opens on the enzyme surface, just above the biphospholipid layer membrane-water interface, where Trp residues are frequently found. In this work, the physicochemical nature of Ca2+ selectivity over Mg2+ on the surface of the SR Ca2+-ATPase has been investigated using the density functional theory (DFT) method. The selection process can be regarded as the first step of the specificity of the enzyme to transport Ca2+. Subsequently, the specificity of the entrance channel to conduct Ca2+ over other cations has also been explored. As revealed by thermodynamic analyses, either the aromatic or the aliphatic amino acid residues distributed on the surface of Ca2+ -ATPase have a bigger affinity to Mg2+ than to Ca2+, resulting in a concentration decrease of free Mg2+ in the local region. Thus, Ca2+ can transport into the Ca2+-entrance channel more easily. Whereafter, for a small quantity of Mg2+ entering this channel accompanying the Ca2+ Z current, the strong electrostatic interactions between Mg2+ and the ligands will limit the activity of this metal ion, which facilitates the weakly bonded Ca2+ passing through the channel at a relatively high rate, as suggested by the "sticky-pore" hypothesis. Furthermore, the corresponding theoretical investigations have demonstrated that Z the increase of the ligand electronegativity can enhance their discrimination between these two cations effectively.