The silver-supporting diatomite was prepared by the vacuum heating decomposition method with diatomite as the carrier and AgNO3 solution as the reaction solution. Its structure was characterized by X-ray diffraction (XRD). The factors influencing silver content of diatomite have been discussed in prepared silver-supporting diatomite. The results show that when the mass ratio of diatomite to AgNO3 is 10:1, the dipping time of 2 hours and the decomposing temperature of 350°C are suitable to preparing silver-supporting diatomite. XRD analyses show that the Ag loaded on the surface and micropore of the diatomite is nano-silver. The antimicrobial effect of silver-supporting diatomite was investigated against Escherichia coli (E. coli). The results show that the silver-supporting diatomite have obvious inhibiting effect on E. coli and its minimum inhibitory concentration decrease with increase of its silver content. The silver-supporting diatomite with the silver content of 1.46% is suitable to kill E. coli in water.