The aim of this research was to determine the antibacterial properties and mechanisms of Cu(II)-exchanged montmorillonite (MMT-Cu) in vitro. Escherichia coli ATCC K88 and Salmonella choleraesuis ATCC 50020 were chosen as indicators of intestinal tract pathogenic bacteria in weanling pigs. The antibacterial activity of MMT-Cu and MMT were evaluated by determining the minimum inhibitory concentrations (MICs) using two-fold serial dilutions in MH broth, and the amount of Cu2+ released into the broth was measured by an atomic absorption technique. The rate of oxygen consumption was measured using a SP-II-type oxygen electrode analyzer; the structural integrity of cell walls of bacteria was observed by transmission electron microscope (TEM); enzymatic activity of bacteria was examined with a semi-automatic biochemical analyzer. The results showed that MMT-Cu inhibited the growth of E. coli K88 and S. choleraesuis, and the MICs were 1024 and 2048 microg/ml, respectively. The amount of Cu2+ released into the broth was in the range 6.51-45.65 microg/ml. Nevertheless, both tested bacteria still grew in broth containing 32,768 microg/ml of MMT. Treatment with MMT-Cu could lead to significant release of intracellular enzymes from the tested bacteria. Data from oxygen consumption of bacteria showed that MMT-Cu could inhibit the TCA pathway of the bacterial respiration metabolism. These results show that MMT-Cu has an antibacterial activity.