Abstract: The aims of this study were to prepare Cu²⁺-loaded montmorillonite (Cu-MMT) and investigate its bactericidal activity and mechanism. Cu-MMT was prepared by the method of ion exchange reaction. The structure and surface characteristic of Cu-MMT were determined. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Cu-MMT against the strain of Escherichia coli were determined. The activities of intracellular enzyme in bacterial solution were measured, and the morphology of E.coli was observed during the interaction between Cu-MMT and bacteria. The results showed that treatment with Cu²⁺ increased cation exchange capacity of montmorillonite, but specific surface area and surface negative charge density were decreased. The MIC and MBC of Cu-MMT against the tested E.coli were 0.16 and 0.64 mg·mL^-1, respectively. Cu-MMT could destroy bacterial cellular membrane and then resulted in leakage of intracellular enzymes such as asparate aminotransferase, lactate dehydrogenase and alanine aminotransferase. These suggest that Cu-MMT has a strong bactericidal activity. The bactericidal mechanism of Cu-MMT may be that bacteria are adsorbed by Cu-MMT, and then morphology and permeability of cellular membrane are changed. This leads to an efflux of intracellular contents and the death of bacteria.