Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture.