Human metallothionein III (MT III) gene was synthesized with Escherichia coli preference codon usage and expressed in E. coli in glutathione-S-transferase (GST) fusion form. The recombinant MT III was released by proteinase Factor Xa digestion and purified with the yield of 2 mg/L culture, and its specific Cd2+ binding capability was confirmed. E. coli strain BL21(DE3), expressing MT III, showed metal tolerance between 0.1 and 0.5 mM Cd2+ and bacterial growth was inhibited at 1 mM Cd2+. MT III expressing E. coli strain showed binding discrimination between different metal ions in combination use, with the preference order of Cd2+ > Cu2+ > Zn2+. It absorbed different metal ions with relatively constant ratio and showed a cumulative absorption capability for mixed heavy metals.