Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins existing as partially metal-depleted species and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. Electrochemistry, mass spectrometry and molecular dynamics approaches unravel the chemical coordination of Zn(II) in all species, and their thermodynamic differences have been elucidated. Finally, we proposed the molecular basis for the origin of the differences in Zn(II) binding affinities to MTs.