Montmorillonite (MMT) and its Cu2+-exchanged montmorillonite (Cu-MMT) were used to study the antibacterial activity on Aeromonas hydrophila. The results indicated that MMT had no antibacterial activity. The minimum inhibitory concentration (MIC) and bactericidal concentration (MBC) of Cu-MMT on A. hydrophila are found to be 150 and 600 mg/L, respectively. The continuance of the antibacterial activity of Cu-MMT was much longer than copper sulfate. In order to reveal the mechanism of the antibacterial activity of Cu-MMT, the Cu release from Cu-MMT in tryptic soy broth (TSB) was investigated. In the first 2 h, Cu concentration in the supernatant reaches saturated value, about 1.22-2.27% of the overall Cu in Cu-MMT suspended in the medium. The washed Cu-MMT in TSB for 24 h retained their full antibacterial activity; whereas, the supernatants from the washed pellets showed very little antibacterial activity. These findings suggested that the antibacterial activity of Cu-MMT was mainly localized on the clay surface, and not due to the release of Cu2+ into solution. The excessive positive charge of Cu-MMT would make Cu-MMT attract A. hydrophila with negatively charged cellular wall. In this case, the copper cation would act directly on the bacteria adsorbed on the surface of Cu-MMT, instead of into the medium. The mechanism for the antibacterial activity of Cu-MMT may involve the enhanced affinity of Cu-MMT for A. hydrophila and the antibacterial activity of Cu2+.